Ferrate(1-), [[N,N'-1,2-ethanediylbis[N-[[2-(hydroxy-.kappa.O)phenyl]methyl]glycinato-.kappa.N,.kappa.O]](4-)]-, sodium (1:1), (OC-6-13)-

Endpoint summary

Description of key information

Fe(Na)HBED is skin sensitizer according to the criteria outlined in the Regulation (EC) 1272/2008, since 69% (instead of trigger 30% and more) of treated animals showed positive skin sensitization response after the challenge exposure.

Justification for selection of skin sensitisation endpoint:
Guideline and GLP study

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14.10.2008 - 5.12.2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

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 2011/20012 before the LLNA-method has become prefered method.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Animals
Males and females of Dankin Hartley guinea pig, coming from conventional
Husbandry of Laboratory Animals in Ilkowice were used in the pilot and main study.
The animals were quarantined and observed daily for 5 days [SPR/T/6]. The
general medical-veterinary examination was performed on day of introduction of animals to quarantine. Detailed medical-veterinary examination was performed prior to the beginning of experiment. Animals without any clinical signs were
introduced to the experiment.
All animals were individually marked.
Two males with the average body weight of 589.5 g (body weight range: 572g
– 607g) and three females with the average body weight of 433.7 g (body weight
range: 369g – 468g) were used in the pilot study. The animals of pilot study were
from 3 to 4 months old. Four 3.5-month-old males with the average body weight of
543.5 g (body weight range: 486g – 574g) and nineteen 3.5-month-old females with
the average body weight of 477.2 g (body weight range: 366g – 534g) were used in
the main study.

Housing conditions
During quarantine and experiment the animals were kept in air-conditioned
room with the following parameters:
- air temperature: 21 – 22 °C
- relative air humidity: 50 – 65%
- artificial fluorescent lighting: 12 hours light – 12 hours dark.
The animals were kept individually in cages with plastic bottom and wired lid, with
dimensions: (length x width x height) 58 x 37 x 21 cm.
UV-sterilized wooden shavings were used as a bedding [SPR/T/16, SPR/T/48].
Each cage was equipped with label containing information on study code, group
mark, experimental starting and ending date, sex and number of animal.

Fodder and water
The animals were given ad libitum standard granulated LSK fodder produced
by Wytwórnia Koncentratów i Mieszanek Paszowych AGROPOL, Motycz and tap
water with supply of ascorbic acid (0.6% solution).

Route:

intradermal

Vehicle:

other: Aqua pro inectione

Concentration / amount:

Injection 1: 1:1 mixture (v/v) FCA / aqua pro injectione
Injection 2: 4% aqueous suspension of test item
Injection 3: 1:1 mixture (v/v) of test item in aqua pro injectione at concentration of
4% with 1:1 mixture (v/v) FCA / aqua pro injectione

Day(s)/duration:

Day 0

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50% was applied in volume about 1 ml to gazue patches (2 x 4 cm)

Day(s)/duration:

48 h

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50% aqueous suspension of test item in volume of 0.5 mL was applied to
gauze patches with dimensions 2 x 2 cm and laid on skin of right flank. The distilled
water (medium) was applied in the same manner on skin of left flank. The gauze
patches were covered with PCV foil and elastic bandage. The exposure time was 24
hours.

Day(s)/duration:

24h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Two males with the average body weight of 589.5 g (body weight range: 572g
– 607g) and three females with the average body weight of 433.7 g (body weight range: 369g – 468g)
were used in the pilot study. The animals of pilot study were
from 3 to 4 months old. Four 3.5-month-old males with the average body weight of
543.5 g (body weight range: 486g – 574g) and nineteen 3.5-month-old females with
the average body weight of 477.2 g (body weight range: 366g – 534g) were used in
the main study.

Details on study design:

Pilot study
The aim of pilot study was to determinate concentrations of test item to be
used in the main study. The test item was mixed with aqua pro injectione in case of
intradermal injections and with distilled water in case of epidermal application.
In order to determine concentrations for I step of induction – intradermal
injections, frontal part of back of two guinea pigs was shaved on the area of about
4.5 x 6 cm and the intradermal injections with 0.1 mL of aqueous suspension of test item were made. The following concentrations were tested: 0.5%, 1%, 2%, 4% and
8%. Observations of skin reactions were performed after 24, 48, 72, 96 and 120
hours since injections. Results of observations are presented in Tables 1 - 2.
Concentration of 4% was chosen for the main study due to its mild to moderate
influence on skin.
In order to determine concentration for II step of induction – topical application
and concentration for challenge – topical application, the flanks of three guinea pigs
were shaved on the area of about 4.5 x 6 cm. The test item at concentrations of 30%,
40% and 50% was 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. Two concentrations were tested on each
guinea pig. The observations of skin reactions were performed after 24, 48, 72, 96
and 120 hours since the end of exposure. Results of observations are presented in
Tables 3-5.
Test item concentration of 50% not causing any skin irritation was chosen for
induction in the main study – topical application as well as for the challenge.
Freunds Complete Adjuvant was given intradermally to all animals of the pilot
study. Two injections with 0.1 mL of 1:1 mixture (v/v) FCA / aqua pro injectione were
given in the shoulder region.

The main study
Induction – intradermal injections
The day before experiment frontal part of back of treated and control animals
was shaved on the area of about 4.5 x 6 cm with the aid of shaver.
Day 0 – treated group
Three pairs of intradermal injections of 0.1 mL were given to prepared skin of
2 males and 14 females so that one of each pair of injection lay on each side of the
midline.
Injection 1: 1:1 mixture (v/v) FCA / aqua pro injectione
Injection 2: 4% aqueous suspension of test item
Injection 3: 1:1 mixture (v/v) of test item in aqua pro injectione at concentration of
4% with 1:1 mixture (v/v) FCA / aqua pro injectione
Day 0 – control group
Three pairs of intradermal injections of 0.1 mL were given to 2 males and 5
females of control group in the same sites as in the treated animals.
Injection 1: 1:1 mixture (v/v) FCA / aqua pro injectione
Injection 2: aqua pro injectione (medium)
Injection 3: 1:1 mixture (v/v) of aqua pro injectione with 1:1 mixture (v/v) FCA / aqua
pro injectione

Induction – topical application
Day 6 – treated and control group
Approximately 24 hours before topical application, frontal part of back of
treated and control animals was shaved in the sites of intradermal injections. In view
of no irritating properties of test item the shaved skin surface was painted with 0.5
cm3 of 10% sodium lauryl sulfate in vaseline, in order to create a local dermatitis.
Day 7 – treated group
The 50% aqueous suspension of test item was applied to skin of animals of
treated group in the sites of intradermal injections. The aqueous suspension of test
item was applied in volume of about 1 mL to multilayered gauze patches with
dimensions 2 x 4 cm and then laid on skin. The gauze patches were covered with
PCV foil and elastic bandage. After 48 hours the band and gauze patches were
removed from skin.
Day 7 – control group
Distilled water was applied on skin of control animals in the sites of intradermal
injections. Manner of application, protection of gauze patches and time of exposure
was the same as in case of treated animals.

Challenge – topical application
Day 20 – treated and control group
Approximately 24 hours prior to the topical application, flanks of treated and
control animals were shaved on the area of about 4.5 x 6 cm.
Day 21 – treated and control group
The 50% aqueous suspension of test item was applied to prepared skin of
right flank of animals of treated and control group.
The 50% aqueous suspension of test item in volume of 0.5 mL was applied to
gauze patches with dimensions 2 x 2 cm and laid on skin of right flank. The distilled
water (medium) was applied in the same manner on skin of left flank. The gauze
patches were covered with PCV foil and elastic bandage. The exposure time was 24
hours. Following this time the band and gauze patches were removed.

Challenge controls:

yes

Positive control substance(s):

yes

Remarks:

Freunds Complete Adjuvant

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50% aqueous suspension of test item

No. with + reactions:

0

Total no. in group:

16

Clinical observations:

no pathological changes were stated on skin of animals of treated group in the site of test item application. No pathological changes on skin were stated also in the site of medium application

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50% aqueous suspension of test item

No. with + reactions:

8

Total no. in group:

16

Clinical observations:

discrete or patchy erythema was stated on skin of seven females; discrete or patchy eryhtema with dryness of epidermis were observed in one female. No pathological changes were stated in the remaining animals.

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

other: 3 rd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

50% aqueous suspension of test item

No. with + reactions:

11

Total no. in group:

16

Clinical observations:

discrete or patchy erythema was stated on treated area of skin of one female; discrete of patchy erythema with dryness of epidermis were observed in ten females.

Remarks on result:

positive indication of skin sensitisation

Remarks:

On the ground of obtained results one may state that allergic skin reactions occurred in 11 animals of treated group, what is 69% of all animals of treated group.

During experiment no changes in behavior were observed in animals of control

and treated group. No clinical signs were stated in animals of both control and treated

group.

One female of control group died during experiment. At necropsy presence of

pink-brown blood clots in chest comprising heart and left side of lungs was stated.

The stated changes evidence for cardiac rupture. This death should be considered

as accidental and not connected with test item because guinea pigs are sensitive

animals which die spontaneously quite often.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

On the ground of the study, the test item Fe (III) HBED may be included to the agents causing sensitization of skin of guinea pigs.

Executive summary:

Skin sensitization study of Fe (III) HBED was performed according to the OECD Guideline for Testing of Chemicals No 406 / Method B.6,

SPR/T/24 as well as Good Laboratory Practice Principles (GLP-OECD, 1997) were methodological basis of the study. The experiment was conducted on guinea pigs according to maximization method of Magnusson and Kligman.

16 animals in treated group and 7 animals in control group were used in the main study. Concentrations of test item used in the main study were determined in the pilot study.

The main study comprised two parts: induction and challenge. Induction was performed in two steps. In the first step the animals were given 4% aqueous suspension of test item with Freunds Complete Adjuvant (FCA). In the second step, the 50% aqueous suspension of test item was applied to skin on sites of intradermal injections. During period of induction group of control animals was subjected to sham treatment – it was given water (medium) instead of test item. In order to challenge sensitization the 50% aqueous suspension of test item (challenge dose) was applied to right flank of treated and control animals. Distilled water (medium) was applied to left flank.

Following induction, skin reaction of treated and control animals was evaluated after 24, 48 and 72 hours since the end of exposure.

During readings allergic skin reactions were stated in 11 animals of treated group. No pathological changes were stated on skin of animals of control group. On the ground of the study one may state that Fe (III) HBED may be included to agents causing sensitization.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitization study is performed in Dunkin-Hartley guinea pigs in order to obtain information on possibility of allergic contact dermatitis, due to contact of Fe(Na)HBED with skin (Mrzyk, 2008). The study was conducted according to the OECD TG 406 without deviations. The experiment was commenced by the pilot study in which concentrations of test item for the main study were established. Concentration of 4% causing mild skin changes was selected to step I of the main experiment (induction - intradermal injections). Concentration of 50% not causing changes on skin was selected for the step II of the main experiment (induction – topical application). Since the test item is solid, the maximum concentration of test item applied to the skin could be 50%. This concentration did not cause changes on skin and therefore was selected for the step III of the main experiment (challenge). 16 animals in treated group and 7 animals in control group were used in the main study. In the step I of the main study, the animals of treated group were given intradermally 4% aqueous solution of test item with Freund’s Complete Adjuvant (FCA). In the step II of the main study, 50% test item in aqueous solution was applied to the skin on the site of intradermal injections. As the 50% aqueous solution of the test item did not cause skin irritation, on day before step II of the experiment, 10% sodium lauryl sulphate in Vaseline was applied on the site of intradermal injections, in order to create local skin irritation (day 6). During period of induction, group of control animals was subjected to sham treatment – they were given medium (water) instead of test item. 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. Following challenge, skin reactions of treated and control animals were evaluated after 24, 48 and 72 hours after the end of exposure. General clinical observations were performed during the main study. Detailed skin observations were conducted after 24, 48 and 72 hours after the end of exposure. 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 the killing of animals.

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

During reading after 24 since the end of exposure, no pathological changes were stated on skin of animals of treated group in the site of test item application. No pathological changes on skin were stated also in the site of medium application.

During reading after 48 hours since the end of exposure, discrete or patchy erythema was stated on skin of seven females; discrete or patchy eryhtema with

dryness of epidermis were observed in one female. No pathological changes were stated in the remaining animals. No pathological changes were stated on skin of all animals in the site of medium application.

During reading after 72 hours since the end of exposure discrete or patchy erythema was stated on treated area of skin of one female; discrete of patchy

erythema with dryness of epidermis were observed in ten females. On the ground of obtained results one may state that allergic skin reactions occurred in 11 animals of treated group, what is 69% of all animals of treated group.

During experiment body weight gain was observed in all animals of control and treated group except for one female in which slight body weight loss occurred. The average body weight gain in males of treated group was greater than in control group, whereas the average body weight gain in females of treated group was slightly lower in comparison with females of control group. During experiment no changes in behavior were observed in animals of control

and treated group. No clinical signs were stated in animals of both control and treated group.

One female of control group died during experiment. At necropsy presence of pink-brown blood clots in chest comprising heart and left side of lungs was stated. The stated changes evidence for cardiac rupture. This death should be considered as accidental and not connected with test item because guinea pigs are sensitive animals which die spontaneously quite often.

Justification for classification or non-classification

According to the CLP criteria, skin sensitization response is considered to be positive if skin reactions were present in at least 30% of the treated animals (in case of an adjuvant guinea pig test method). In the available skin sensitization study with Fe(Na)HBED, 69% of animals were sensitized at the second reading time point (72-hour from the start of exposure). Therefore, the test material meets criteria for classification and labelling as skin sensitizer according to the Regulation (EC) No 1272/2008.