Zinc EDDHA

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 (CIBA-GEIGY, 1993). 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 (BASF, 2010). 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.

For the source substance ZnSO₄ two LLNA studies are available (Basketter et al. 1999, Ikarashi et al. 1992). In both studies Zn was not considered to possess the ability to cause skin sensitisation in humans, and, thus, would not be classified and labeled as sensitiser.

Based on these results the target substance is not considered to bear a skin sensitising potential.

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:

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

Adequacy of study:

key study

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Parameter:

SI

Value:

1.6

Test group / Remarks:

5%

Parameter:

SI

Value:

2.9

Test group / Remarks:

10%

Parameter:

SI

Value:

2.96

Test group / Remarks:

25%

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.

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.
The same result is expected for the organic constituents of target substance since it has the same composition as the source substance.

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:

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

Adequacy of study:

key study

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

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

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

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

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

Group:

positive control

Remarks on result:

not measured/tested

Remarks:

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

The animals gained in body weight.

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. Since only slight/barely perceptible reactions were observed at 48-hour reading point after challenge exposure, the substance is considered to have very weak sensitisation potential.
The same result is expected for the organic constituents of target substance since it has the same composition as the source substance.

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.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Parameter:

SI

Value:

2.3

Test group / Remarks:

25 % dose group

Parameter:

SI

Value:

2

Test group / Remarks:

10 % dose group

Parameter:

SI

Value:

1.3

Test group / Remarks:

5 % dose group

Cellular proliferation data / Observations:

Zinc did not stimulate lymph node cell proliferation at least threefold greater than that observed in concurrent vehicle-treated control and therefore was judged to be negative. With Zn, there was a slight suggestion of a dose-response trend, but even at 25% (the highest concentration at which it was possible to test), the stimulation index reached only 2.3 (< 3), and so it was recorded as negative.

Interpretation of results:

GHS criteria not met

Remarks:

EU GHS criteria not met

Conclusions:

Zn was not considered to possess, to any significant degree, the ability to cause skin sensitisation in humans, and, thus, would not be classified and labeled as sensitiser. Thus, the Zn ions of the target substance are also expected to be not sensitising.

Executive summary:

The LLNA was used to determine the skin sensitization potential of 13 metal salts. With Zn, there was a slight suggestion of a dose-response trend, but even at 25% (the highest concentration at which it was possible to test), the stimulation index reached only 2.3, and so it was recorded as negative. In conclusion, Zn was not considered to possess the ability to cause skin sensitisation in humans, and, thus, would not be classified and labeled as sensitiser.

Thus, the Zn ions of the target substance are also expected to be not sensitising.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Parameter:

SI

Remarks:

zinc sulphate

Value:

1.41

Test group / Remarks:

concentration 1: 10 %

Parameter:

other: disintegrations per minute (DPM)

Remarks:

zinc sulphate

Value:

0.002

Variability:

0.00077 (standard deviation)

Remarks on result:

other: Mean counts per minute

Parameter:

SI

Value:

2.32

Test group / Remarks:

positive control

Parameter:

SI

Test group / Remarks:

concentration 2

Remarks on result:

not measured/tested

Parameter:

SI

Test group / Remarks:

concentration 3

Remarks on result:

not measured/tested

Interpretation of results:

GHS criteria not met

Remarks:

EU GHS criteria not met

Conclusions:

The stimulation index was determined to be 1.41 (mean cpm is even below the mean cpm of the vehicle). Therefore, Zn is not sensitising. Thus, the Zn ions of the target substance are also expected to be not sensitising.

Executive summary:

In a dermal sensitisation study with Zinc sulphate heptahydrate in 20% ethanol solution, 6-8 week old female Balb/c mice (three animals / dose) were tested in the murine local lymph node assay. The test substance was applied on three consecutive days on the dorsum of each ear, local lymph nodes were isolated on fourth day after application. Isolated cells were cultivated with [3H]methyl thymidine ([3H]TdR) and [3H]TdR incorporation was measured with a scintillation counter. The stimulation index (SI) for ZnSO₄ was determined to be 1.41 (SI < 2), showing no significant increase in lymphocyte proliferation compared to vehicle alone. In this study, ZnSO₄ is not a dermal sensitiser. This study is considered to be reliable and to fulfill general scientific requirements.

Thus, the Zn ions of the target substance are also expected to be not sensitising.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Fe(Na)EDDHA

There are two studies at hand that evaluate skin sensitisation 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 sensitising 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 sensitisation (needed for haptenisation).

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

Justification for classification or non-classification

In conclusion, considering all available information the registered substance is not classified with regard to sensitisation in accordance with Regulation (EC) No 1272/2008.