Sodium [[α,α'-(ethylenediimino)bis[2-hydroxybenzene-1-acetato]](4-)]ferrate(1-)

Endpoint summary

Description of key information

In the GPMT with the source substance Fe(Na)EDDHA, 20% and 55% of the animals of the test group showed skin reactions 24 and 48 hours after removing the dressings, respectively. The study concludes that the test substance FeNaEDDHA has, therefore, a skin sensitisation potential. With only slight/barely perceptible reactions needing for a major part 48 hrs to develop, based on a challenge concentration that is only half of what is needed to induce irritation in the animals in this study, the sensitising potential can only concluded to be very minimal at the most. Additionally, LLNA conducted with the source substance Fe(Na)EDDHA resulted in a SI of 2.96 for the highest concentration of 25% tested. The results can be considered ambiguous as both the conclusions non-sensiting and sensiting are equally justified. With the a maximum SI of about 3.0, no difference in response was observed between 10% concentration and the highest possible concentration of 25%. This indicates a very weak sensitising effect at the most. Additionally, these results could be mediated by an accidentally low response of the control group.

In conclusion, considering all available information, o,o-Fe(Na)EDDHA is not classified with regard to sensitisation in accordance with Regulation (EC) No 1272/2008 (CLP).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-04-13 to 2010-04-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 20.3 +/- 1.1 g
- Housing: single in Makrolon Type II, with wire mesh top cages
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 degree C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 6.00 a.m. - 6.00 p.m.

Vehicle:

dimethylformamide

Concentration:

5, 10, and 25% (w/w) in dimethylformamide
Application volume: 25 µL

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which can be technically used, was a 25% (w/w) solution in dimethylformamide.
- Irritation:
To determine the highest non-irritant test item concentration that does at the same time not induce systemic toxicity, a pre-test was performed in two animals. Two mice were treated by topical (epidermal) application to the dorsal surface of each ear with test item at concentrations of 10% or 25% once daily each on three consecutive days. Clinical signs were recorded within 1 hour and 24 +/- 4 hours after each application as well as on day 6. Furthermore, prior to the first application of the test item and before sacrifice the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). Additionally, for both animals, the ears were punched after sacrifice at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm²) and were immediately pooled per animal and weighed using an analytical balance.
At the tested concentrations the animals did not show any signs of systemic toxicity. Also, the animals did not show relevant signs of local irritation as confirmed by the ear thickness and ear weight measurements.


MAIN STUDY
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10, and 25% (w/w) in dimethylformamide. The application volume, 25 µL, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the vehicle alone (control animals).
Five days after the first topical application, all mice were administered with 250 µL of 81.7 µCi/mL 3HTdR (corresponds to 20.4 µCi 3HTdR per mouse) by intravenous injection
via a tail vein.
For the determination of lymph node weights, after excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. After excision of the lymph nodes, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm²). For each animal both punches were immediately weighed per animal using an analytical balance. Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Sodium. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared. The level of 3HTdR incorporation was then measured on β -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute
(DPM).
- Criteria used to consider a positive response:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

In addition to the sensitising reactions the following observations were recorded during the test and observation period: Mortality / Viability, Body weights, Ear thickness, Ear weights, Lymph node weights, Clinical signs (local / systemic).

Positive control substance(s):

other: no positive control

Statistics:

The mean values and standard deviations were calculated in the body weight tables. The ANOVA (Dunnett-test) was conducted on the ear and lymph node weights to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. However, both biological and statistical significance were considered together.

Parameter:

SI

Remarks on result:

other: Stimulation Indices (S.I.) of 1.6, 2.9, and 2.96 were determined with the test item at concentrations of 5, 10, and 25% in dimethylformamide, respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: A statistically significant increase in DPM/animal and in lymph node weights was observed in all test item treated groups in comparison to the vehicle control group (p=0.008). A dose response was obtained.

Clinical signs, mortality and body weights:

During the course of the study, the animals did not show any signs of systemic toxicity and no cases of mortality were observed. The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age. Although reddening of the ears could not be assessed due to the intensive colour of the test item, other signs of local irritation (e.g. ear swelling) were also not seen. A statistically significant increase in ear weights was not observed in the test item treated groups in comparison to the vehicle control group.

Table 1: Calculation and results of individual data

Test item concentration % (w/w)	Group number	Animal number	DPM values measured	DPM-BG per animal (2 lymph nodes)^a)	S.I.^b)
—	BG I	—	16	—
—	BG II	—	15	—
0	1	1	488	473	—
0	1	2	1199	1187	—
0	1	3	569	554	—
0	1	4	716	701	—
0	1	5	916	901
5	2	6	887	872	1.1
5	2	7	1375	1360	1.8
5	2	8	1361	1346	1.8
5	2	9	1073	1058	1.4
5	2	10	1428	1413	1.9
10	3	11	3017	3002	3.9
10	3	12	2387	2372	3.1
10	3	13	1367	1352	1.8
10	3	14	3106	3091	4.1
10	3	15	1198	1183	1.6
25	4	16	2256	2241	2.9
25	4	17	2393	2378	3.1
25	4	18	2454	2439	3.2
25	4	19	1269	1254	1.6
25	4	20	3090	3075	4.0

BG = Background (1 mL 5% trichloroacetic acid) in duplicate;

1 = Vehicle Control Group for the Test Item (dimethylformamide);

2-4 = Test Groups S.I. = Stimulation Index;

a) = Values corrected for mean background value (BGI and BGII);

b) = Stimulation Indices relative to the mean of the control group (Group 1)

Table 2: Calculation of Stimulation Indices per dose group

Test item concentration	Mean DPM per Group (5 animals)^a)	SD^b)	S.I.
Vehicle for the Test Item (dimethylformamide)	762.1	286.3	1.0
5 % acetic acid, oxo-, sodium salt	1209.3	234.3^c)	1.6
10 % acetic acid, oxo-, sodium salt	2199.5	897.2^c)	2.9
25 % acetic acid, oxo-, sodium salt	2276.9	655.9^c)	3.0

Table 3: Calculation of the EC3 value

	Test item concentration%	S.I.
Test Group3	10(a)	2.9(b)
Test Group4	25(c)	3.0(d)
EC3=(a-c) [(3-d)/(b-d)]+c=25 %(w/w)

EC3 = Estimated concentration for a S.I. of 3.

Interpretation of results:

ambiguous

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item FeNaEDDHA resulted to a SI of 2.96 under the conditions of this study for the highest concentration of 25% tested. The results can be considered ambiguous as both the conclusions non-sensiting and sensiting with EC3 value of 25% (w/w) are equally justified.

Executive summary:

In order to study a possible skin sensitization potential of FeNaEDDHA, three groups each of five female mice were treated daily with the test item at concentrations of 5, 10, and 25% (w/w) in dimethylformamide by topical application to the dorsum of each ear (left and right) once daily for three consecutive days. A control group of five mice was treated with the vehicle (dimethylformamide) only. 25% was the highest technically achievable concentration suitable for application which did at the same time not cause excessive skin irritation or systemic toxicity as confirmed by a pre-experiment. The test was performed according to the OECD Guideline 429 and the Commission Directive 2004/73/EC B.42. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in ab-scintillation counter.

Results of the formulation analysis showed that the measured concentrations of FeNaEDDHA were within the acceptable range set at 100 +/- 15% of the nominal concentration. The obtained results ranged between 100.8 – 104.5% of the nominal values. The test item was found to be stable in all dose formulation samples for 2 hours at room temperature when protected from light.

During the course of the study, the animals did not show any signs of systemic toxicity and no cases of mortality were observed. Although reddening of the ears could not be assessed due to the intensive colour of the test item, other signs of local irritation (e.g. ear swelling) were also not seen. A statistically significant increase in ear weights was not observed in the test item treated groups in comparison to the vehicle control group.

In this study Stimulation Indices of 1.6, 2.9, and 2.96 were determined with the test item at concentrations of 5, 10, and 25% in dimethylformamide, respectively. A statistically significant increase in DPM/animal and in lymph node weights was observed in all test item treated groups in comparison to the vehicle control group (p=0.008). A dose response was obtained.

The test item FeNaEDDHA resulted to a SI of 2.96 under the conditions of this study for the highest concentration of 25% tested. The results can be considered ambiguous as both the conclusions non-sensiting and sensiting with EC3 value of 25% (w/w) are equally justified.

Some remarks can be made to this study:

- With a maximum SI of 2.96 at the highest dose, it indicates a very weak sensitizing effect at the most. Also the notion that there is no difference in response between 10% and highest possible concentration of 25%.

- Notably the DPM of the non treated controls of the test group is much lower than that of the non-treated controls of the positive control group. In case these animals are treated exactly similarly (same size and age animals and same amount of radio-labelled thymidine injected and same timing of evaluations) one would expect similar levels. When considering the possibility that control group shows accidentally a too low activity, the study would be evaluated as negative.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-12-23 to 1994-01-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The in vitro study existed already before the LLNA method was adopted as the method of first choice.

Species:

guinea pig

Strain:

other: Pirbright White Strain

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY Limited, Animal Production, 4332 Stein, Switzerland
- Weight at study initiation: 326 to 424 g
- Housing: individually in Macrolon cages (Type 3)
- Diet (e.g. ad libitum): ad libitum, standard guinea pig pellets - NAFAG No. 845
- Water (e.g. ad libitum): ad libitum, fresh water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 degree C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hrs light cycle

Route:

intradermal and epicutaneous

Vehicle:

physiological saline

Concentration / amount:

Intradermal: 5% test substance in physiological saline
Epicutaneous: 50% test substance in vaseline
Challenge: 10% test test substance in vaseline

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Intradermal: 5% test substance in physiological saline
Epicutaneous: 50% test substance in vaseline
Challenge: 10% test test substance in vaseline

No. of animals per dose:

10 male/ 10 female

Details on study design:

RANGE FINDING TESTS:
The concentration for the intradermal injections was selected on account of the solubility of the test article in standard vehicles and its local and systemic tolerability in a pretest. Since 5% test substance in physiological saline could be injected and was well tolerated, this concentration was used for the intradermal induction.
The concentration for the epidermal applications were selected on account of the primary irritation potential of the test article. The following concentrations of the test item have been examined on separate animals for the determination of the maximum subirritant concentration: 10, 20, 30 and 50% in vaseline. 50% in vaseline was the highest applicable concentration of the test article. Erythema reactions were observed with 20, 30 and 50% test article in vaseline.

MAIN STUDY
A. INDUCTION EXPOSURE
Day 0: Induction, intradermal injections:
3 pair of intradermal onjections (0.1 mL per injection) were made simultaneously into the left and right side of the shaved neck of the test and control group animals.
Test group:
- adjuvant/saline mixture 1:1 (v/v)
- 5% test substance in phys. saline (w/v)
- 5% test substance in the adjuvant/saline mixture (w/v)
Control group:
- adjuvant/saline mixture 1:1 (v/v)
- adjuvant/saline mixture 1:1 (v/v)
- physiol. saline

Day 8: Induction, epidermal application
In the test group test substance was incorporated in vaseline (w/v) and applied on a filter paper patch to the neck of the animals (patch 2x4 cm; approx. 0.4 g per patch; occluded administration for 48 hrs). The control group was treated with the vehicle only.
Test group:
- 50% test substance in vaseline
Control group:
- vaseline only

Day 21: Challenge
The test and control animals were tested on one flank with the test substance in vaseline (w/w) and on the other flank with the vehicle alone (patch 2x2 cm; approx. 0.2 g per patch; occluded for 24 hrs).
Test and control group:
- 10% test substance in vaseline
- vaseline only

The test was performed on 10 male and 10 female guinea pigs in the test group and 5 males and 5 females in the control group, respectively.

Challenge controls:

The test and control animals were tested on one flank with 10% test substance in vaseline (w/w) and on the other flank with vaseline alone (patch 2x2 cm; approx. 0.2 g per patch; occluded for 24 hrs).

Positive control substance(s):

no

Remarks:

the sensitivity of strain is checked once or twice a year with a known mild or moderate sensitiser: mercaptobenzothiazole, hexyl cinnamic aldehyde, or potassiumdichromate.

Positive control results:

The animal test strain was tested with 5% of 2-mercaptobenzothiazole (Test No. 930012) in Oleum arachidis (intradermal induction); 50% in vaseline (epidermal induction) and 30% in vaseline (challenge) (Test period: July 5, 1993- August 5, 1993). 90% of animals showed positive reactions 24 and 48 hours after challenge exposure.

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10% test substance in vaseline

No. with + reactions:

4

Total no. in group:

20

Clinical observations:

erythema, edema

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10% test substance in vaseline. No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: erythema, edema.

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

10% test substance in vaseline

No. with + reactions:

11

Total no. in group:

20

Clinical observations:

erythema, edema

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 10% test substance in vaseline. No with. + reactions: 11.0. Total no. in groups: 20.0. Clinical observations: erythema, edema.

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

10% test substance in vaseline

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10% test substance in vaseline. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

10% test substance in vaseline

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 10% test substance in vaseline. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.

The animals gained in body weight.

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the experimental conditions employed, 20% and 55% of the animals of the test group showed skin reactions 24 and 48 hours after removing the dressings, respectively. The test substance is, therefore, classified as a mild to moderate sensitiser in albino guinea pigs according to the grading of Magnusson and Klingman.

Executive summary:

A sensitisation test in albino guinea pigs was performed to determine the contact allergenic potency of the test article in albino guinea pigs. This test was based on the OECD Guideline No. 406 (Skin Sensitisation) and the EU method B.5 (Skin Sensitisation).

On day 0 three pairs of intradermal injections (0.1 mL per injection) were made simultaneously into the left and right side of the shaved neck of the test and control group animals.

On day 8 test substance was incorporated in vaseline (w/v) and applied on a filter paper patch to the neck of the animals (patch 2x4 cm; approx. 0.4 g per patch; occluded administration for 48 hrs) in the test group. The control group was treated with the vehicle only.

On day 21 (Challenge) the test and control animals were tested on one flank with the test substance in vaseline (w/w) and on the other flank with the vehicle alone (patch 2x2 cm; approx. 0.2 g per patch; occluded for 24 hrs).

Under the experimental conditions employed, 20% and 55% of the animals of the test group showed skin reactions 24 and 48 hours after removing the dressings, respectively. The test substance is, therefore, classified as a mild to moderate sensitiser in albino guinea pigs according to the grading of Magnusson and Klingman.

There are some remarks that can be made to this study:

- Unfortunately there are no observations reported of the skin reactions following the intra- and epi-dermal applications during induction.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There are two studies at hand that evaluate skin sensitization potential of the structural analogue Fe(Na)EDDHA (CAS 84539-55-9):

The skin sensitisation potential of the source substance Fe(Na)EDDHA was examined in the Maximisation Test of Magnusson and Kligman (GPMT) in guinea pigs according to OECD Guideline 406 (CIBA-GEIGY, 1994; Report No. 931145). Under the experimental conditions employed, 20 and 55% of the animals of the test group showed skin reactions 24 and 48 hours after removing the dressings, respectively. Unfortunately, there are no observations reported of the skin reactions following the intra- and epi-dermal applications during induction. All observed positive reactions for erythema and oedema were ''very slight/barely perceptible". This offers a possibility for subjective readings. It is not indicated whether evaluations of the individual animals were performed in a blinded way. With only slight/barely perceptible reactions needing for a major part 48 h to develop, based on a challenge concentration that is only half of what is needed to induce irritation in the animals in this study, the sensitizing potential can only concluded to be very minimal at the most. The test substance was considered therefore to have, if any, a very weak skin sensitisation potential in the GPMT. 

The source substance UVCB Fe(Na)EDDHA has also been tested in a LLNA, in which a stimulation index (SI) of 1.6, 2.9, and 2.96 were determined at concentrations of 5, 10 and 25 % in dimethylformamide, respectively. A statistically significant increase in DPM/animal and in lymph node weights was observed in all treated groups in comparison to the vehicle control group (p=0.008) (BASF, 2010a). A dose response was also present. However, also in this study, with the results showing a maximum SI of about 3.0 there was no difference in response between 10% concentration and the highest possible concentration of 25%. Additionally, these results could be mediated by an accidentally low response of the control group.

Therefore, based on these study results both conclusions (sensitising and not sensitising) can be made.

Methylated structural analogues FeEDDHMANa (CAS 84539-53-7) and FeEDDHMAK (EC 405-420-1) are very similar in their physical/chemical properties (high water solubility, low Kow, no hydrolysis in water, no reactivity to protein). These analogues can therefore be expected to behave very similar with respect to dermal penetration and subsequent availability in viable dermis and protein reactivity needed for sensitisation. However, various studies have not indicated any sensitizing potential for these analogues (please refer to read-across statement).

In addition it is noted that FeNaEDDHA does not interact with protein, which is considered a requirement for sensitization (needed for haptenisation).

Finally, also decades of use of this substance did not result to any reports of people becoming sensitized.

Justification for classification or non-classification

Two available studies indicate either a possible weak, or an ambiguous result for a weak potential for sensitisation of the source substance FeNaEDDHA. Structural analogues and other comparable chelates are not sensitising. The substance does not interact with proteins and decades of use of this substance did not result to any reports of people sensitised to it. Based on the structural and chemical similarity the same ambiguous result could be expected for the target substance o,o-Fe(Na)EDDHA, if it was tested in a sensitisation study.

In conclusion, considering all available information, o,o-Fe(Na)EDDHA is not classified with regard to sensitisation in accordance with Regulation (EC) No 1272/2008 (CLP).