Sodium long chain alkyl salicylate

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:

20 August 2014 to 29 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

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:

TEST ANIMALS
- Strain: CBA/J strain, inbred, SPF-Quality
- Age at study initiation: Approximately 10 to 11 weeks old
- Weight at study initiation: 19 to 24 g. Body weight variation was within ± 20 % of the sex mean.
- Housing: During acclimation, animals were group housed in cages (height 18 cm) containing sterilised sawdust as bedding material. Paper and shelters (disposable paper corner home) were supplied as cage-enrichment. Prior to the study, the animals were group housed with a sheet of paper instead of sawdust and cage enrichment.
- Diet: Free access to pelleted rodent diet
- Water: ad libitum access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: at least 10 air changes/hour
- Photoperiod: 12-hour light/12-hour dark cycle

Vehicle:

methyl ethyl ketone

Concentration:

2.5, 5 and 10 % w/w

No. of animals per dose:

5 animals per dose

Details on study design:

RANGE FINDING TEST
A pre-screen test was conducted in order to select the highest test material concentration to be used in the main study. Initially, two test material concentrations were tested; 25 and 50 % concentration.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (5 and 10 %).

MAIN STUDY
INDUCTION - DAYS 1, 2 AND 3
The dorsal surface of both ears was topically treated (25 µL/ear) with the test material concentration. 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 material.

EXCISION OF THE NODES - DAY 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 µCi of ³H-methyl thymidine. After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of 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
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) for each animal 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 200 x g for 10 minutes at 4 °C. To precipitate the DNA, the LNC were treated with 5 % trichloroacetic acid (TCA) and stored in the refrigerator until the next day.

RADIOACTIVITY MEASUREMENTS - DAY 7
Precipitates were recovered by centrifugation, re-suspended 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).

OBSERVATIONS
- Mortality/Viability: Twice daily
- Body weights: Day 1 (pre-dose) and Day 6 (prior to necropsy)
- Clinical signs: Once daily on Days 1 to 6 (on Days 1 to 3 between 3 and 4 hours after dosing)
- Irritation: Once daily on Days 1 to 6 (on Days 1 to 3 within 1 hour after dosing) according to the numerical scoring system below. Furthermore, a description of all other (local) effects was recorded.
- Necropsy performed: No

Grading for Irritation Reactions:
Erythema and eschar formation:
0 = No erythema
1 = Very slight erythema (barely perceptible)
2 = Well-defined erythema
3 = Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth)
4 = Severe erythema (beet redness) to eschar formation preventing grading of erythema

Positive control substance(s):

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

Statistics:

Although the data do not permit the use of linear interpolation, calculation of EC3 values (the estimated test material concentration that will give an SI =3) by loglinear extrapolation can provide a reliable estimation of sensitisation potency class for use in risk assessment and can avoid the need for repeat animal testing. The data show a clear dose-response and a slope ratio of 0.175 was calculated indicating that extrapolation can provide a reliable estimation of the EC3 value according to Ryan et al. 2007. An estimated EC3 value of 0.2 % was calculated.

Positive control results:

A reliability test with three concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v) was performed several months prior to the study using the same materials, animal supplier, animal strain and essential procedures as for the main test. For both scientific and animal welfare reasons, no concurrent positive control group was added to the study.
The positive control was tested at concentrations of 0, 5, 10 and 25 %. The SI values calculated were 1.2, 1.4 and 4.7, respectively. An EC3 value of 17.3 % was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5 %. Based on the results, it was concluded that the LLNA as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.

Parameter:

SI

Value:

>= 9.9

Test group / Remarks:

2.5% concentration

Parameter:

SI

Value:

>= 11.9

Test group / Remarks:

5% concentration test group

Parameter:

SI

Value:

>= 16.3

Test group / Remarks:

10% concentraiton test group

Pre-screen test

Very slight to well-defined erythema was noted for the animals treated with 25 and 50 % concentrations between Days 2 and 4.

Beige staining of test material remnants and/or scaliness on the dorsal surface of the ears of these animals on Days 5 and 6 did not hamper scoring of erythema. Variations in ear thickness during the observation period were more than 25 % from Day 1 pre-dose values, although measurements were difficult to conduct since the ears were hardened by test material remnants. Ptosis was noted for the animals on Day 3. Eschar formation was seen on the right ear of animal 1 on Day 6. A wound was seen on the right ear of animal 2 on Day 6.

At a 5 and 10 % test material concentration, no signs of systemic toxicity were noted; no irritation was observed and variations in ear thickness during the observation period were less than 25 % from Day 1 pre-dose values.

Based on these results, the highest test material concentration selected for the main study was 10

 

Main study

- Skin reactions / Irritation

No irritation of the ears was observed in any of the animals examined. A dull left eye was noted for one animal treated at 5 % on Days 1 and 6.

- Systemic toxicity

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

- Macroscopy of the auricular lymph nodes and surrounding area

All auricular lymph nodes of the animals of the experimental groups were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Table 1: Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI)

Test Material Concentration (% w/w)	Animal Number	Size of Nodes¹		DPM²/animal	Mean DPM ± SEM³	Mean SI ± SEM
		Left	Right
0	1	N	N	493	546 ± 86	1.0 ± 0.2
	2	N	N	635
	3	N	N	825
	4	N	N	459
	5	N	N	318
2.5	6	++	++	6297	5429 ± 1016	9.9 ± 2.4
	7	+	+	3309
	8	++	++	4511
	9	++	++	8982
	10	++	++	4045
5	11	++	++	7717	6509 ± 455	11.9 ± 2.1
	12	++	++	5967
	13	++	+	5535
	14	++	++	5826
	15	++	++	7499
10	16	++	++	12 114	8876 ± 1120	16.3 ± 3.3
	17	++	++	6572
	18	++	++	7007
	19	++	++	7709
	20	++	++	10 980

¹Relative size of auricular lymph nodes. N = normal; + or ++ = degree of enlargement

²DPM = Disintegrations per minute

³SEM = Standard error of the mean

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test material was determined to be sensitising (category 1A).

Executive summary:

An assessment of contact hypersensitivity to the test material was made in the mouse. A Local Lymph Node Assay (LLNA) study was carried out in accordance with the standardised guidelines OECD 429, EU Method B.42 and EPA OPPTS 870.2600 under GLP conditions.

Test material 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 material concentrations of 2.5, 5 or 10 % w/w in methyl ethyl ketone on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone.

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.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

All auricular lymph nodes of the animals of the experimental groups were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with the test material at concentrations of 2.5, 5 and 10 % were 5429, 6509 and 8876 DPM, respectively. The mean DPM/animal value for the vehicle control group was 546 DPM. The SI values calculated for the test material concentrations 2.5, 5 and 10 % were 9.9, 11.9 and 16.3, respectively. The data show a clear dose response and a slope ratio of 0.175 was calculated indicating that extrapolation can provide a reliable estimation of the EC3 value. An estimated EC3 value of 0.2% was calculated.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.

Under the conditions of this study the test material was determined to be sensitising (category 1A).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

An assessment of contact hypersensitivity to the test material was made in the mouse. A Local Lymph Node Assay (LLNA) study was carried out in accordance with the standardised guidelines OECD 429, EU Method B.42 and EPA OPPTS 870.2600 under GLP conditions.

Test material 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 material concentrations of 2.5, 5 or 10 % w/w in methyl ethyl ketone on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone.

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.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

All auricular lymph nodes of the animals of the experimental groups were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with the test material at concentrations of 2.5, 5 and 10 % were 5429, 6509 and 8876 DPM, respectively. The mean DPM/animal value for the vehicle control group was 546 DPM. The SI values calculated for the test material concentrations 2.5, 5 and 10 % were 9.9, 11.9 and 16.3, respectively. The data show a clear dose response and a slope ratio of 0.175 was calculated indicating that extrapolation can provide a reliable estimation of the EC3 value. An estimated EC3 value of 0.2% was calculated.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.

Under the conditions of this study the test material was determined to be sensitising (category 1A).

Migrated from Short description of key information:
Sensitising (LLNA); OECD 429, EU Method B.42 and EPA OPPTS 870.2600

Justification for selection of skin sensitisation endpoint:
Only one study available.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The SI values calculated for the test material concentrations 2.5, 5 and 10 % were 9.9, 11.9 and 16.3, respectively. The data show a clear dose response and a slope ratio of 0.175 was calculated indicating that extrapolation can provide a reliable estimation of the EC3 value. An estimated EC3 value of 0.2% was calculated. Substances with an EC3 value less than or equal to 2% are classified in Category 1A.

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance requires classification with respect to skin sensitisation as Category 1A (H317: may cause an allergic skin reaction).