Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and hydroxybenzenesulfonic acid monosodium salt, iron sodium salts

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2000-08-10 to 2000-09-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Directive No. 96/54/EEC, B.6, 30 July 1996

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was conducted prior to the adoption of OECD 429 for LLNA.

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, 76410 Saint-Aubin-les-Elbeuf, France
- Age at study initiation: 1-3 months
- Weight at study initiation: 375 ± 24 g for the males and 368 ± 17 g for the females
- Housing: individually in polycarbonate cages (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle
- Diet (e.g. ad libitum): ad libitum ("106 pelleted diet" (UAR, 91360 Villemoisson-sur-Orge, France))
- Water (e.g. ad libitum): ad libitum (filtered by a FG Millipore membrane (0.22 micron))
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 30 to 70
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: To:

Route:

intradermal and epicutaneous

Vehicle:

physiological saline

Remarks:

0.9% NaCl, batch No. 2831 (Laboratoire Frésénius, 92316 Sèvres, France)

Concentration / amount:

Preliminary test:
- By intradermal route (tested concentrations: 50%, 25% and 10% (w/w));
- By cutaneous route (tested concentrations: 50%, 25%, 10% and 5% (w/w)).

Main test:
Induction phase:
- By intradermal route: 10% (w/w);
- By cutaneous route: 10% (w/w).
Challenge phase:
- 5% and 50% (w/w)

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Remarks:

0.9% NaCl, batch No. 2831 (Laboratoire Frésénius, 92316 Sèvres, France)

Concentration / amount:

Preliminary test:
- By intradermal route (tested concentrations: 50%, 25% and 10% (w/w));
- By cutaneous route (tested concentrations: 50%, 25%, 10% and 5% (w/w)).

Main test:
Induction phase:
- By intradermal route: 10% (w/w);
- By cutaneous route: 10% (w/w).
Challenge phase:
- 5% and 50% (w/w)

No. of animals per dose:

- two males and two females for the preliminary test;
- 30 animals (15 males and 15 females) for the main test: a control group of ten animals (five males and five females) and a treated group of 20 animals (ten males and ten females).

Details on study design:

RANGE FINDING TESTS:
A preliminary test was conducted in order to determine the concentrations to be tested in the main study.
By intradermal route:
- 24 hours before treatment, the dorsal region of the animals was clipped,
- intradermal administrations of the dosage form preparations (0.1 mL) were performed in the interscapular region,
- cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections.

By cutaneous route:
- 24 hours before treatment, both flank regions of the animals were clipped,
- the filter paper of a chamber (Finn Chamber ) was fully-loaded with the dosage form preparations. The chamber was then applied to the clipped area of the skin (one concentration per flank). The chamber was held in place by means of an occlusive dressing for 24 hours,
- cutaneous reactions were evaluated approximately 24 and 48 hours after removal of the dressings.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (on day 1: intradermally; on day 8: epicutaneous)

- Test groups:
Intradermal injections (day 1): six injections (0.1 mL) were made (three pairs) into the left and right side of a 4 cm x 2 cm clipped interscapular area:
1) 50% Freund's complete adjuvant (v/v) and sterile isotonic solution (0.9% NaCl);
2) test substance at 10% (w/w) in 0.9% NaCl;
3) test substance at 10% (w/w) in a mixture of FCA/0.9% NaCl (50/50);
Cutaneous application (day 7): 0.5 mL of sodium lauryl sulfate at 10% (w/w) in vaseline in order to induce local irritation;
Cutaneous application (day 8): test substance at 10% (w/w) in 0.9% NaCl

- Control group:
Intradermal injections (day 1): six injections (0.1 mL) were made (three pairs) into the left and right side of a 4 cm x 2 cm clipped interscapular area:
1) 50% Freund's complete adjuvant (v/v) and sterile isotonic solution (0.9% NaCl);
2) 0.9% NaCl;
3) 0.9% NaCl (w/v) in a mixture of FCA/0.9% NaCl (50/50);
Cutaneous application (day 7): 0.5 mL of sodium lauryl sulfate at 10% (w/w) in vaseline in order to induce local irritation;
Cutaneous application (day 8): 0.9% NaCl

By cutaneous application (day 8), a pad of filter paper (approximately 8 cm²) was fully-loaded with the test substance or vehicle alone and then applied to the interscapular region of animals. The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster).

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 22
- Exposure period: 24 h occlusively
- Test groups: left flank: test substance at 5%; right flank: test substance at 50%
- Control group: left flank: test substance at 5%; right flank: test substance at 50%
- Site: posterior left and right flanks
- Concentrations: 5% and 50% (w/w)
- Evaluation (hr after challenge): 48 hours

OTHER:

Challenge controls:

On day 22, the animals of treated and control groups received an application of the test substance and vehicle. The filter paper of a chamber (Finn Chamber®) was fully-loaded with the test substance at concentrations of 5% and 50% (w/w) and was then applied to a clipped area of the skin of the posterior left and right flanks of all animals, respectively.
The chambers were held in contact with the skin for 24 hours by means of an adhesive anallergenic waterproof plaster.

Positive control substance(s):

yes

Remarks:

mercaptobenzothiazole

Positive control results:

One control group of 5 animals and one treated group of 10 animals received the positive control substance at the concentration of 1% (w/w) intradermally on day 1; 20% (w/w) was applied cutaneously on day 8
On day 22, MERCAPTOBENZOTHIAZOLE at the concentration of 20% (w/w) induced positive skin sensitization reactions in 70% guinea-pigs (CIT/Study No. 20068 TSG - July 2000).

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

5%

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

brown coloration of the skin which could have masked a possible moderate erythema

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 5%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: brown coloration of the skin which could have masked a possible moderate erythema.

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

brown coloration of the skin which could have masked a possible moderate erythema: 7 animals; a possible intense erythema: 3 animals

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: brown coloration of the skin which could have masked a possible moderate erythema: 7 animals; a possible intense erythema: 3 animals.

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

5%

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

no coloration of the skin: 1 animal; no coloration/or brown coloration of the skin: 5 animals; brown coloration of the skin which could have masked a possible discrete erythema: 4 animals

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 5%. No with. + reactions: 9.0. Total no. in groups: 10.0. Clinical observations: no coloration of the skin: 1 animal; no coloration/or brown coloration of the skin: 5 animals; brown coloration of the skin which could have masked a possible discrete erythema: 4 animals.

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

50%

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

brown coloration of the skin: 1 animal; brown coloration of the skin which could have masked a possible discrete erythema: 2 animals; a possible moderate erythema: 6 animals; a possible intense erythema: 1 animal

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 50%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: brown coloration of the skin: 1 animal; brown coloration of the skin which could have masked a possible discrete erythema: 2 animals; a possible moderate erythema: 6 animals; a possible intense erythema: 1 animal.

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

brown coloration of the skin which could have masked a possible moderate erythema: 18 animals; a possible intense erythema: 2 animals

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5%. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: brown coloration of the skin which could have masked a possible moderate erythema: 18 animals; a possible intense erythema: 2 animals.

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

brown coloration of the skin which could have masked a possible moderate erythema: 13 animals; a possible intense erythema: 7 animals

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: brown coloration of the skin which could have masked a possible moderate erythema: 13 animals; a possible intense erythema: 7 animals.

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

5%

No. with + reactions:

16

Total no. in group:

20

Clinical observations:

no coloration of the skin: 4 animals; no coloration/or brown coloration of the skin: 5 animals; brown coloration of the skin which could have masked a possible discrete erythema: 8 animals; a possible moderate erythema: 3 animals

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 5%. No with. + reactions: 16.0. Total no. in groups: 20.0. Clinical observations: no coloration of the skin: 4 animals; no coloration/or brown coloration of the skin: 5 animals; brown coloration of the skin which could have masked a possible discrete erythema: 8 animals; a possible moderate erythema: 3 animals.

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

50%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

no/or brown coloration of the skin: 2 animals; brown coloration of the skin which could have masked a possible discrete erythema: 3 animals; a possible moderate erythema: 15 animals

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 50%. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: no/or brown coloration of the skin: 2 animals; brown coloration of the skin which could have masked a possible discrete erythema: 3 animals; a possible moderate erythema: 15 animals.

No clinical signs and no deaths were observed during the study. The body weight gain of the treated animals was similar to that of the control animals.

Challenge phase - Scoring of cutaneous reactions

On the left flank receiving the test substance at the concentration of 5%

A brown coloration of the skin, which could have masked a possible moderate or intense erythema (grade 2 or 3), was observed at the 24-hour reading in all animals of the control and treated groups. At the 48-hour reading, a brown coloration of the skin, which could have masked a possible discrete or moderate erythema (grade 1 or 2), persisted in 4/10 animals of the control group and in 11/20 animals of the treated group; dryness of the skin was also recorded in 1/20 animals of the treated group. No cutaneous reactions were observed in the other animals of both groups.

On the right flank receiving the test substance at the concentration of 50%

A brown coloration of the skin, which could have masked a possible moderate or intense erythema (grade 2 or 3), was observed at the 24-hour reading in all animals of the control and treated groups. At the 48-hour reading, no cutaneous reactions were observed in 1/10 animals of the control group and in 2/20 animals of the treated group. A brown coloration of the skin, which could have masked a possible discrete to intense erythema (grades 1 to 3), persisted in 9/10 animals of the control group and in 18/20 animals of the treated group.

Microscopic examination

Control group: right flank receiving the test substance at the concentration of 50%

Four out of five control males and all the control females showed minimal to moderate acanthosis in the right flank. This was associated in all of them with slight inflammatory cell infiltration of dermis (mainly mononuclear cells) and in some of them with minimal hyperkeratosis, minimal intercellular edema in epidermis, minimal mononuclear cell infiltration in epidermis and/or hair follicle(s) and /or minimal extravasation of red blood cells in the dermis. All these findings were considered to be due to skin irritation.

Treated group: right flank receiving the test substance at the concentration of 50%

Minimal to moderate acanthosis, together with slight inflammatory cell infiltration in the dermis (mainly mononuclear cells), were observed in all the treated males and females. For some of them, minimal hyperkeratosis, minimal intercellular edema in epidermis, minimal mononuclear cell infiltration in epidermis and/or hair follicle(s), minimal extravasation of red blood cells in the dermis and/or vascular ectasia in the upper dermis were also noted. All these findings had similar morphological characteristics as those observed in the males and females from the control group. Moreover, their severity was also similar and none of them showed severity higher than that observed in the control group of animals treated with the test substance at the concentration of 50%. Thus, all the findings observed on the right flanks of all the treated males and females were considered to be due to skin irritation but not to a sensitization process.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance EDDHAS Fe 3K does not induce delayed contact hypersensitivity in guinea pigs.

Executive summary:

The potential of Fe3KEDDHSA to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman (CIT, 2001; OECD TG 406). Thirty guinea pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females. The concentration of 10% was determined in a preliminary study and the vehicle used was 0.9% NaCl. On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals: Freund's complete adjuvant (FCA) diluted at 50% (v/v) with vehicle (both groups), test substance in vehicle (treated group) or vehicle alone (control group), test substance in a mixture FCA/0.9% NaCl 50/50 (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl 50/50 (control group). On day 7, the same region received a topical application of sodium lauryl sulfate in vaseline (10%, w/w) in order to induce local irritation. On day 8, the test substance (treated group) or the vehicle (control group) was applied topically to the same test site, which was then covered by an occlusive dressing for 48 hours. On day 22, all animals of the treated and control groups were challenged by a cutaneous application of the test substance to the left and right flank at concentrations of 5% and 50% (w/w), respectively. Test substance was maintained under an occlusive dressing for 24 hours. Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing. At the end of the study, animals were killed without examination of internal organs. Skin samples were taken from the challenge application sites of all the animals. A histopathological examination was performed on the preserved skin samples from the right flank (test site receiving the test substance at the maximal practicable concentration of 50% (w/w) in sterile isotonic saline solution (0.9% NaCl)).

No clinical signs and no deaths were noted during the study. After the challenge application by 5% of the test substance, a brown coloration of the skin, which could have masked a possible moderate or intense erythema, was observed at the 24-hour reading in all animals of the control and treated groups. At the 48-hour reading, a brown coloration of the skin, which could have masked a possible discrete or moderate erythema, persisted in 4/10 animals of the control group and in 11/20 animals of the treated group; dryness of the skin was also recorded in 1/20 animals of the treated group. No cutaneous reactions were observed in the other animals of both groups. On the right flank receiving the test substance at the concentration of 50% a brown coloration of the skin, which could have masked a possible moderate or intense erythema, was observed at the 24-hour reading in all animals of the control and treated groups. At the 48-hour reading, no cutaneous reactions were observed in 1/10 animals of the control group and in 2/20 animals of the treated group. A brown coloration of the skin, which could have masked a possible discrete to intense erythema, persisted in 9/10 animals of the control group and in 18/20 animals of the treated group. The microscopic examinations performed on the skin samples from the right flank receiving the test substance at the concentration of 50% revealed no evidence of a sensitization process. In conclusion, the test substance Fe3KEDDHSA does not induce delayed contact hypersensitivity in guinea pigs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The potential of Fe(3K)EDDHSA (EC No. 462-490-6) to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman (Centre International de Toxicologie, 2001a; OECD Guideline 406).Thirty guinea pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females. The concentration of 10% was determined in a preliminary study and the vehicle used was 0.9% NaCl. On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals: Freund's complete adjuvant (FCA) diluted at 50% (v/v) with vehicle (both groups), test substance in vehicle (treated group) or vehicle alone (control group), test substance in a mixture FCA/0.9% NaCl 50/50 (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl 50/50 (control group). On day 7, the same region received a topical application of sodium lauryl sulphate in Vaseline (10%, w/w) in order to induce local irritation. On day 8, the test substance (treated group) or the vehicle (control group) was applied topically to the same test site, which was then covered by an occlusive dressing for 48 hours. On day 22, all animals of the treated and control groups were challenged by a cutaneous application of the test substance to the left and right flank at concentrations of 5% and 50% (w/w), respectively. Test substance was maintained under an occlusive dressing for 24 hours. Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing. At the end of the study, animals were killed without examination of internal organs. Skin samples were taken from the challenge application sites of all the animals. A histopathological examination was performed on the preserved skin samples from the right flank (test site receiving the test substance at the maximal practicable concentration of 50% (w/w) in sterile isotonic saline solution (0.9% NaCl)).

No clinical signs and no deaths were noted during the study. After the challenge application by 5% of the test substance, a brown coloration of the skin, which could have masked a possible moderate or intense erythema, was observed at the 24-hour reading in all animals of the control and treated groups. At the 48-hour reading, a brown coloration of the skin, which could have masked a possible discrete or moderate erythema, persisted in 4/10 animals of the control group and in 11/20 animals of the treated group; dryness of the skin was also recorded in 1/20 animals of the treated group. No cutaneous reactions were observed in the other animals of both groups. On the right flank receiving the test substance at the concentration of 50% a brown coloration of the skin, which could have masked a possible moderate or intense erythema, was observed at the 24-hour reading in all animals of the control and treated groups. At the 48-hour reading, no cutaneous reactions were observed in 1/10 animals of the control group and in 2/20 animals of the treated group. A brown coloration of the skin, which could have masked a possible discrete to intense erythema, persisted in 9/10 animals of the control group and in 18/20 animals of the treated group. The microscopic examinations performed on the skin samples from the right flank receiving the test substance at the concentration of 50% revealed no evidence of a sensitization process. In conclusion, the test substance Fe(3K)EDDHSA does not induce delayed contact hypersensitivity in guinea pig.

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

The skin sensitisation potential of 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. The test substance was considered therefore to have, if any, a very weak sensitisation potential in the GPMT. However, the skin reactions at 48 -h reading were only slight/barely perceptible reactions and were based on a challenge concentration that is only half of what was needed to induce irritation in the animals. Based on these study results, both conclusions can be made: sensitising and not sensitising.

In a Local Lymph Node Assay (LLNA), skin sensitization potential of Fe(Na)EDDHA was determined according to OECD Guideline 429 and Commission Directive 2004/73/EC B.42 (BASF SE, 2010, Report No. 1324900). In this study, Stimulation Indices 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). A dose response was present.

The results for the test item Fe(Na)EDDHA was found to be ambiguous for skin sensitiser under the test conditions of this study and an EC3 value of 25 % (w/w) was derived. 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%. Therefore, this indicates a very weak sensitizing potential at the most.

In addition it is noted that Fe(3Na)EDDHSA does not interact with proteins (please refer to the profiling results of the OECD QSAR Toolbox included in the read-across justification), 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.

Migrated from Short description of key information:
The nearest neighbour Fe(3K)EDDHSA (EC No. 462-490-6) tested in a Guinea Pig Maximisation Test was negative. All skin reactions observed in the control and in the treated groups were considered to be due to skin irritation but not to a sensitisation process. The other closely related substance Fe(Na)EDDHA (CAS No. 84539-55-9) induced very minimal sensitisation response in the same Test (OECD 406). In a LLNA, Fe(Na)EDDHA resulted in a SI of 2.96 for the highest concentration of 25% tested (LLNA) (OECD 429). The results can be considered ambiguous as both the conclusions non-sensiting and sensiting with EC3 value of 25% (w/w) can equally be justified. Additionally, these results could be mediated by an accidentally low response of the control group. Furthermore, methylated chelates Fe(Na)EDDHMA (CAS 84539-53-7) and Fe(K)EDDHMA are not skin sensitizers in Guinea Pig Maximisation Test. Based on these results, the target substance Fe(3Na)EDDHSA does not possess skin sensitisation potential.

Justification for selection of skin sensitisation endpoint:
Key study conducted with the nearest analogue Fe(3K)EDDHSA (EC 462-490-6)

Justification for classification or non-classification

Based on the result of the skin sensitisation study, available for the nearest neighbour Fe(3K)EDDHSA (EC No. 462 -490 -6), the target substance Fe(3Na)EDDHSA is not subject to classification and labelling for skin sensitisation according to Regulation (EC) 1272/2008.