Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and reported GLP study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J strain, inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

Number of animals: 20 females (nulliparous and non-pregnant), five females per group.
Age and body weight: young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.
Identification: tail mark with marker pen.

Conditions: environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation: animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: free access to tap water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures.

Vehicle:

other: 1% Pluronic © L92 in Elix water.

Concentration:

10, 25 and 50%

No. of animals per dose:

5

Details on study design:

Induction (days 1,2,3): the dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.

Excision of the nodes (day 6): each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity (day 6): a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and stored in the refrigerator until the next day.

Radioactivity measurements (day 7): precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not used

Positive control results:

The SI values calculated for the HCA concentrations 5, 10 and 25% were 1.7, 1.7 and 4.7 respectively. An EC3 value of 16.5% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.9, 16.0, 11.9, 16.9, 14.4 and 12.8%.
Based on these results it was concluded that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 10, 25 and 50% were 1.2, 1.0 and 1.5, respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 499, 423 and 630 DPM respectively. The mean DPM/animal value for the vehicle control group was 415 DPM.

Table: Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI)

group	TS^1 (%)	animal	Size nodes^2		DPM^3/ animal	mean DPM ±^4			mean SI ± SEM
			left	right
1	0	1	n	n	200	415	±	112	1.0	±	0.4
		2	n	n	430
		3	n	n	826
		4	n	n	391
		5	n	n	228
2	10	6	n	n	751	499	±	68	1.2	±	0.4
		7	n	n	385
		8	n	n	383
		9	n	n	463
		10	n	n	512
3	25	11	n	n	341	423	±	42	1.0	±	0.3
		12	n	n	385
		13	n	n	373
		14	n	n	434
		15	n	n	580
4	50	16	n	n	366	630	±	235	1.5	±	0.7
		17	n	n	1548
		18	n	n	316
		19	n	n	596
		20	n	n	325

1. TS = test substance (% w/w).

2. Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

3. DPM= Disintegrations per minute

4. SEM = Standard Error of the Mean

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

DTPA-FeNaH was considered to be a non skin sensitizer

Executive summary:

The study was carried out based on the guidelines described in: OECD, Section 4, Health Effects, No.429 (2010); EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay"; EPA, OPPTS 870.2600 (2003) “Skin Sensitization”. Test substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (1% L92).  

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No irritation of the ears was observed in any of the animals examined. Yellow/brown test substance remnants were present on the dorsal surface of the ears of both animals at 10, 25 and 50% (Days 1-3), which did not hamper scoring of the skin reactions. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 499, 423 and 630 DPM respectively. The mean DPM/animal value for the vehicle control group was 415 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.2, 1.0 and 1.5 respectively. Since there was no indication that the test substance elicited an SI≥3 when tested up to 50%,DTPA-FeHNa was considered to be a non skin sensitizer

The six-month reliability check withAlpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity. 

Based on these results, DTPA-FeHNa would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and theRegulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The results of this study are in compliance with studies with other metal chelates (see also read across document in section 13).

Migrated from Short description of key information:

In a LLNA (OECD 429), DTPA-FeHNa was not a skin sensitizer.

Justification for selection of skin sensitisation endpoint:

Well performed and reported GLP study

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Based on the knowledge that all known respiratory sensitizers have also tested positive in a skin sensitization test, it is not expected that DTPA-FeHNa would show respiratory sensitizing properties.

Justification for classification or non-classification

Based on the results of the LLNA, no classification needed for skin sensitizing properties.