Butanedioic acid, sulfo-, monoesters with lanolin alcs., disodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 9 to September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Butanedioic acid, sulfo-, monoesters with lanolin alcs., disodium salts

EC Number:

290-844-8

EC Name:

Butanedioic acid, sulfo-, monoesters with lanolin alcs., disodium salts

Cas Number:

90268-43-2

Molecular formula:

The substance is too complex, no molecular formula is available.

IUPAC Name:

Reaction product of lanolin with maleic anhydride and Sodium sulphate.

Test material form:

semi-solid (amorphous): gel

Remarks:

paste

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JN

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A, Calco (Lecco), Italy (Charles River France Laboratories)
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: 16 to 25 grams
- Housing: polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm and nesting material
Cage tray control daily and changed as necessary (at least twice/week)
- Number of animals/cage: 1 mouse at cage during the study; up to 5 during acclimatisation
- Diet: 4 RF 21 from Mucedola S.r.l, Milan, Italy, ad libitum throughout the study
- Water: drinking water supplied to each cage via a water bottle, ad libitum
Record of analyses of water and diet were done. Components present in the drinking water or diet are not a level likely to interfere with the purpose or conduct of the study.
- Acclimation period: at least 5 days
Veterinary health check after arrival. Permanent ink marking on the tail. Animals were identified by odd numbers.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 2 °C
- Humidity (%): 55 % +/- 15 %
- Air changes (per hr): 15 to 20 air changes per hours
- Photoperiod (hrs dark / hrs light): daily light/dark cycle of 12/12 hours with artificial (fluorescent tubes)
Actual conditions were monitored and recorded. No relevant deviations occurred.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Remarks:

preliminary phase: 100, 50, 25, 10, 5% w/w; Main phase: 50, 25, 10 % w/w

Concentration:

TEST ITEM
Preliminary irritancy test: dose volume of 25 µL/ear/day (50 µL/animal/day) at 5-10-25-50-100 % w/w of dose level in term of test item supplied
Main phase: dose volume of 25 µL/ear/day (50 µL/animal/day) at 10-25-50 % w/w of dose level in term of test item supplied
5-bomo-2-deoxyuridine (BrdU)
10 mg/mL in physiological saline (0.9 % NaCl) at the dose volume of 0.5 mL/animal.

No. of animals per dose:

Preliminary irritancy test: 1 animal per dose (marked with odd number).
Main phase: 2 animals per dose (marked with odd number).

Selection/allocation was made randomly. Animals without observable skin lesion were chosen.

Details on study design:

a) DOSING:
PRELIMINARY IRRITANCY TEST:
The doses were applied for three consecutive days (1,2,3) with the test item formulations, by topical application to the dorsal surface of each ear, using a Gilson micropipette.
MAIN STUDY
The doses were applied for three consecutive days (1,2,3) with the test item formulations, by topical application to the dorsal surface of each ear, using a Gilson micropipette. After one day of rest, at 5th day BrdU (5-bromo-2-deoxyuridine) was applied by intraperitoneal injection, using a 25 G needle and a plastic graded syringe of a suitable volume.
At the end mice were sacrificed, processing of lymph nodes and determination of Cell proliferation.
b) IN LIFE OBSERVATION
PRELIMINARY IRRITANCY TEST:
Mortality and morbidity: twice daily
Clinical signs: before dosing on day 1, and daily after dosing commenced (approximately 1 hour after daily dosing, when applicable);
Body weight: at allocation (day 1) and on sacrifice (day 6)
Scoring of dermal reaction: daily, (once before first dosing, before dosing on Days 2 and 3 and daily thereafter).
Irritation to the skin was assigned a numerical value (table 1).
Ear thickness measurement: on Day 1 (before dosing), on Day 3 (before dosing) and on Day 6, ear thickness was measured by a suitable micrometer.
In the Main assay, the test item used at the higher concentrations that are systemically tolerated and judged not to cause excessive reaction at the treatment site. In particular, concentrations are considered irritant if:
-erythema grading (score) is ≥ 3 and/or
-ear thickness is ≥ 25% with respect to Day 1
-ear punch weight is ≥ 25% with reference to the negative control group at any day of measurement
MAIN ASSAY
Mortality and morbidity: twice daily
Clinical signs: before dosing commenced and daily up to sacrifice (approximately 1 hour after each dosing, in the days of treatment).
Body weight: at allocation (day 1) and on sacrifice (day 6)
c) TERMINAL STUDIES
PRELIMINARY IRRITANCY TEST:
Terminal studies: at day 6
Euthanasia method: carbon dioxide narcosis
Necropsy: No necropsy was performed. After sacrifice, regularly shaped biopsies were obtained from both ears and weighed together.
MAIN ASSAY
Terminal studies: at day 6, approximately 24 hours after BrdU injection
Euthanasia method: carbon dioxide narcosis.
Necropsy: no necropsy was performed. Shortly after sacrifice of each animal, the auricular lymph nodes were rapidly excised, pooled on individual basis and individually collected in a solution of 2% BSA-PBS (2% bovine serum albumine (BSA) in phosphate buffered saline, PBS).
Cell suspensions were prepared for the evaluation of proliferation at lymph node was determined.
d) DETERMINATION OF CELLULAR PROLIFERATION
Preparation of single cell suspension: A single cell suspension of lymph node cells (LNC) was prepared from each animal by gentle mechanical disaggregation and passage through a 70 µm nylon mesh. The suspensions thus obtained were centrifuged and the supernatant resuspended in 20 mL of 2% BSA-PBS.
Measurement of BrdU content in lymphocytes DNA: BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001), according to manufacturer instructions.
Briefly, 100 μL of the LNC suspension was added to the wells of a flat-bottom 96-well microplate in triplicate. After fixation and denaturation of the LNC, 100 μL of anti-BrdU antibody labelled with peroxidase was added to each well and allowed to react.
Subsequently the anti-BrdU antibody was removed by washing and 100 μL of the substrate solution was then added and allowed to produce chromogen.
The reaction was finally stopped by adding 25 μL of Stop solution (1 M H2SO4). Absorbance (OD) was detected at 450 nm (with reference wavelength: 690 nm).
e) INTERPRETATION OF DATA
Calculation: BrdU labelling index:
(OD450 – OD BLANK450) – (OD690 – OD BLANK690)
The BrdU labelling index was calculated for each mouse and a group mean was subsequently calculated.

The test item is considered to induce sensitisation when the SI for any single treatment dose group is > = 1.6.
It is not required that an increased response is observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).
Assay validity criteria: The Stimulation Index (SI) of the positive control group is higher than 2.0.

Positive control substance(s):

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

Statistics:

Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be in-homogeneous a Modified test (Cochran and Cox) was applied.
The high dose group only (50% w/w) was significant at statistical analysis.

Results and discussion

Positive control results:

In the group treated with the positive control item, a stimulation index (SI) of 4.42 was calculated. As it was greater than 2, the test system was regarded as valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.84

Test group / Remarks:

Test group - high dose

Results 2

Parameter:

SI

Value:

1.52

Test group / Remarks:

Test group - medium dose

Results 3

Parameter:

SI

Value:

0.95

Test group / Remarks:

Test group -low dose

Cellular proliferation data / Observations:

Stimulation indices (SI) 0.95, 1.52 and 1.84, respectively at low, mid and high dose levels.

Any other information on results incl. tables

PRELIMINARY TEST (table 1, 3, 5)

Five concentrations (100, 50, 25, 10, 5% w/w) were selected to be used in the preliminary phase.

No toxicity signs (clinical signs or toxicologically relevant body weight losses) were observed at any of the tested concentrations.

The evaluation of visible reactions was as follows:

-Very slight erythema (score of 1) was observed at the concentration of 100%;

-Desquamation and/or hardening of the treated ears were observed at the concentration of 100%;

-No erythema was recorded at the concentrations of 50, 25, 10 and 5% w/w.

The evaluation of ear thickness was not considered in the evaluation of the dosage selection, due to the persistency of the test item over the ears which created a rigid layer that masked the real ear thickness.

The evaluation of ear punch weight indicated that the reaction was acceptable (increased less than 25% with respect to the negative control) at all the investigated concentrations.

According to the results described above, the highest concentration selected for the main phase was 50% w/w.

MAIN ASSAY- IN VIVO PHASE (table 2, 4)

No mortality or significant clinical signs were recorded in animals treated at all dose levels.

Body weight decreases/reduced body weight gains observed in some animals from all groups (including controls) were considered of low entity and/or incidental and thus, not toxicologically relevant.

Applicant's summary and conclusion

Conclusions:

The potential of the test item, Sulfosuccinate of Lanolin Alcohol, to cause skin sensitisation reactions following topical application to the skin of CBA/JN (CBA/J) mice, was assessed using the LLNA: BrdU-ELISA method, according to the OECD Guideline for testing of chemicals no. 442b.

A selection dose was done by a preliminary study.
In the main sensitisation assay, the test item was administered at the concentrations of 50, 25, and 10% w/w.
Slight dose-related increases in cell proliferation of draining lymph nodes were observed in the 2 higher treatment groups, being significant at statistical analysis only at the high concentration (50%).
The calculated stimulation indices (SI) were 0.95, 1.52 and 1.84, respectively at low, mid- and high dose levels.
The results obtained in this study indicate that the test item may elicit a mild sensitisation response in mice following dermal exposure, since in at least the high dose group the stimulation index was increased to 1.84. This value, although comprised between 1.6 and 1.9, was statistically significant in comparison with controls.
European Directives concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC) No. 1272/2008 and subsequent revisions) would indicate the following:
Classification : Category 1
Signal word : Warning
Hazard statement: H317: May cause an allergic skin reaction

Executive summary:

The potential of the test item, Sulfosuccinate of Lanolin Alcohol, to cause skin sensitisation reactions following topical application to the skin of CBA/JN(CBA/J) mice, was assessed using the LLNA: BrdU-ELISA method, according to the OECD Guideline for testing of chemicals no. 442b.

Preliminary phase

The undiluted test item and four concentrations (50, 25, 10, 5% w/w) in propylene glycol were selected to be used in the preliminary phase, in order to identify a non-toxic and minimally irritant concentration and avoid false positive results.

No signs of toxicity (toxicologically relevant clinical signs or body weight losses) were observed at the tested concentrations.

According to the results of the irritation screening, the concentration judged as minimally irritant was 50% w/w.

Main assay

In the main assay, the test item was topically administered at the concentrations of 50, 25 and 10% w/w, in propylene glycol.

No mortality or significant clinical signs were recorded in any animal.

Changes in body weight observed during the study were within the expected range for this strain and age of animals.

Slight dose-related increases in cell proliferation of draining lymph nodes were observed in the 2 higher treatment groups, being significant at statistical analysis only at the high concentration (50%). The calculated stimulation indices (SI) were 0.95, 1.52 and 1.84, respectively at low, mid- and high dose levels.

The results obtained in this study indicate that the test item may elicit a mild sensitisation response in mice following dermal exposure, since in at least the high dose group the stimulation index was increased to 1.84. This value, although comprised between 1.6 and 1.9, was statistically significant in comparison with controls.

European Directives concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC) No. 1272/2008 and subsequent revisions) would indicate the following:

Classification: Category 1

Signal word: Warning

Hazard statement: H317: May cause an allergic skin reaction