bis[C11-14-(branched and linear)-alkyl]aminium nonadecaoxohexatungstate

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:

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

Adequacy of study:

key study

Study period:

20-12-2011 to 12-3-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted according to OECD Guideline under GLP conditions.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Species: Mouse, CBA/J strain, inbred, SPF-Quality

Source: Janvier, Le Genest-Saint-Isle, France

Number of animals: 20 females (nulliparous and non-pregnant), five females per group.

Age and body weight: Young adult animals (approx. 9 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.

Identification: Tail mark with marker pen.

Health inspection: A health inspection was performed prior to treatment, to ensure that the animals were in a good state of health.

Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0 degrees Centigrade (actual range: 16.1 – 21.7 degrees Centigrade), a relative humidity of 40-70% (actual range: 38 - 74%) and 12 hours artificial fluorescent light and 12 hours darkness per day.

Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximumlevel of 70% for the relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

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.

Vehicle:

methyl ethyl ketone

Concentration:

0, 0.25, 0.5, 1.0 (test substance; % w/w)

No. of animals per dose:

5

Details on study design:

Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test.

One group of 5 animals was treated with vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 microL/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 microCi 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 4 degrees Centigrade. 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:

A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to the DPM/vehicle control group.

Positive control results:

mean
Group % Hexylcinnamicaldehyde DPM ± SEM SI ± SEM

1 0% 133± 38 1.0 ± 0.4
2 5% 181± 55 1.4 ± 0.6
3 10% 269± 48 2.0 ± 0.7
4 25% 988± 142 7.4 ± 2.4

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The SI values calculated for the substance concentrations 0.25, 0.5 and 1% were 6.1, 17.6 and 29.8 respectively. These results show that the test substance elicits an SI greater than 3. The EC3 value (the estimated test substance concentrationthat will give a SI = 3) was established to be between 0 and 0.25%.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 0.25, 0.5 and 1% were 3290, 9443 and 15946 DPM respectively. The mean DPM/animal value for the vehicle control group was 536.

Pre-screen test

No irritation and no signs of systemic toxicity were observed in any of the animals at 0.5% and 1%. Variations in ear thickness in these animals during the observation period were less than 25% from Day 1 pre-dose values. In animals at 2.5% and higher maximum grade 2 erythema was scored and variations in ear thickness were larger than 25% from Day 1 pre-dose values.

Based on the ear thickness results, the highest test substance concentration selected for the main study was a 1% concentration.

Main study

Skin reactions/Irritation

No irritation of the ears was observed in any of the animals examined.

Systemic toxicity

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Bodyweights and bodyweight gain of experimental animals remained in the same range as controls over the study period. The bodyweight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

Macroscopy of the auricular lymph nodes and surrounding area

All lymph nodes of the experimental groups were larger compared to normal. The largest auricular lymph nodes were found in the higher dose groups.

No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In an OECD 429 study ("Skin Sensitisation: Local Lymph Node Assay"), conducted according to GLP, reaction mass of bis(C11-14-alkyl, branched and linear)amine nonadecaoxo hexatungstate (the surrogate substance) is a sensitiser (Notox B.V., 2012).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In an OECD 429 study ("Skin Sensitisation: Local Lymph Node Assay"), conducted according to GLP, reaction mass of bis(C11-14-alkyl, branched and linear)amine nonadecaoxo hexatungstate (the surrogate substance) is a sensitiser (Notox B.V., 2012).

Migrated from Short description of key information:
Bis[C11-14-(branched and linear)-alkyl]aminium nonadecaoxohexatungstate (EC 700-718-0), based upon results from the surrogate substance is a skin sensitiser.

Justification for selection of skin sensitisation endpoint:
Study completed to OECD Guideline and GLP.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation:

The result obtained from the LLNA-study show that the test substance elicits an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 0.25%. According to criteria as defined in Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures the substance is considered as a Skin Sensitiser Category 1A.