Registration Dossier

Administrative data

Description of key information

No positive reactions were observed in the key Guinea pig maximisation test for registered substance and in the Local Lymph Node assay in mice with read-across substance butanedioic acid, sulfo-, 4-C16-18 (even numbered)-alkyl ester(CAS 90268 -39 -6). In vitro testing was waived based on pre-existing in vivo studies. Based on these results, the Registered substance is not regarded as a skin sensitizer according to the recommendations made in the test guidelines.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Oct 2014 to 05 Dec 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
May 2008, including most recent amendments
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000; including the most recent partial revisions
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Sulfosuccinates may cause false positive/negative results in the LLNA, therefore the GPMT is a golden standard test for these compounds.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: approx. 4-8 weeks old
- Weight at study initiation: 361-477 g
- Housing: Group housing of maximally 5 animals per cage
- Diet: Complete maintenance diet for guinea pigs (SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 21 Oct 2014 To 05 Dec 2014
Route:
intradermal
Vehicle:
water
Concentration / amount:
Preliminary test:
Intradermal injection: 40%, 20%, 10%, 5%, 2%, 1%, 0.5%, 0.2%.
Epidermal exposure: 40%, 20%, 10%, 5%.

Main study:
Intradermal injection: 0.5%
Epidermal application: 10%
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Preliminary test:
Intradermal injection: 40%, 20%, 10%, 5%, 2%, 1%, 0.5%, 0.2%.
Epidermal exposure: 40%, 20%, 10%, 5%.

Main study:
Intradermal injection: 0.5%
Epidermal application: 10%
No. of animals per dose:
10 (test group)
5 (control group)
Details on study design:
RANGE FINDING TESTS:
For the intradermal injections a series of four test substance concentrations was tested; the highest concentration was the maximum concentration that could technically be injected. Two animals received two different concentrations in duplicate (0.1 mL/ site) in the clipped scapular region. The resulting dermal reactions were assessed 24 and 48 hours after treatment. Based on the results in the initially treated animals, two additional animals were treated in a similar manner with four lower concentrations at a later stage.
For the epidermal application, a series of four test substance concentrations was tested, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal semi-occlusively to the clipped flank (site of 2x3cm). The animals receiving intradermal injections were treated with the lowest concentrations and two other animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test substance using water. The treated skin areas were assessed for irritation 24 and 48 hours after exposure.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: one intradermal injection on day 1; epidermal application on day 8
- Exposure period (epidermal application): 24 hours
- Test group: 10 animals
- Control group:5 animals
- Site: scapular area
- Concentrations: 0.5% (intradermal injection), 10% (epidermal application)

B. CHALLENGE EXPOSURE
- No. of exposures: single
- Day of challenge:day 22
- Exposure period: 24 hours
- Test groups: 10% test substance
- Control group: vehicle only
- Site: flank
- Evaluation (hr after challenge): at 24 and 48 hours after removal of the dressing
Challenge controls:
Historical control data are provided.
Positive control substance(s):
yes
Remarks:
Alpha- hexylcinnamaldehyde
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20% Alpha- hexylcinnamaldehyde
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
The skin reactions observed in nine experimental animals in response to the 20% test substance concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals.
Remarks on result:
positive indication of skin sensitisation
Remarks:
90% sensitisation rate in reliability check performed in July/August 2014
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
20% Alpha- hexylcinnamaldehyde
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
The skin reactions observed in nine experimental animals in response to the 20% test substance concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals.
Remarks on result:
positive indication of skin sensitisation
Remarks:
90% sensitisation rate in reliability check performed in July/August 2014

No mortality occurred and no symptoms 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.

On day 3 after intradermal injection of 1:1 Mixture of FCA and water (and a 1:1 Mixture of FCA and vehicle for injection), all animals had erythema grade 3. One control animal had slight erythema (grade 1) related to injection of vehicle. This reaction was also found in 6 guinea pigs of the test group after injection with 0.5% test substance. Additionally, one animal in the test group had well-defined erythema (grade 2). One animal from the test group had signs of necrosis (1 mm in diameter). Signs of necrosis were seen in all animals of the test group at the injection site of 1:1 Mixture of FCA and a 20% test substance concentration (size ranging form 3 to 5 mm, on average 3.8 mm); No skin reaction was seen after epidermal exposure to the vehicle or to 10% test substance in any of the animals.

Interpretation of results:
not sensitising
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Based on the results of a guinea pig maximisation test, performed according to OECD/EC guidelines and GLP principles, disodium isodecyl sulfosuccinate was found to be not sensitising.
Executive summary:

A guinea pig maximisation test was performed with disodium isodecyl sulfosuccinate according to OECD/EC guidelines and GLP principles. Reliable positive and negative controls were included. Based on a preliminary irritation study, the test substance concentrations selected for the main study were a 0.5% concentration for the intradermal induction and a 10% concentration for the epidermal induction exposure. A 10% test substance concentration was selected for the challenge phase. During the main study, no mortality occurred and no symptoms of systemic toxicity were observed. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Epidermal exposure to the vehicle or to 10% test substance did not result in a skin reaction in any of the animals. Since no responses were observed in the experimental animals in response to a 10% test substance concentration in the challenge phase, it is concluded that disodium isodecyl sulfosuccinate has no sensitising properties according to CLP Regulation (EC) No. 1272/2008.

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:
See attached read-across justification
Reason / purpose:
read-across source
Species:
mouse
Strain:
CBA:J
Remarks:
inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Not provided.
- Age at study initiation: Young adult animals (approx. 10 weeks old) were selected.
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). 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. 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 (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: at least 5 days before the start of treatment, under laboratory conditions
- Indication of any skin lesions: Health inspection was performed at least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (range daily mean 21.8-22.5°C)
- Humidity (%): 40 to 70% (range daily mean 47-75%)
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
- IN-LIFE DATES: From: 26 August 2016 To: 05 October 2016
Vehicle:
propylene glycol
Remarks:
Merck, Darmstadt, Germany
Concentration:
PRE-SCREEN TEST 25 or 50% w/w
MAIN TEST: 10, 25 or 50% w/w
No. of animals per dose:
PRE-SCREEN TEST: 2
MAIN TEST: 5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility:
The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at Charles River Den Bosch. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). There was no information available regarding the solubility or stability in vehicle.
- Irritation:
Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing). See also Erythema Scores.
In addition a description of all other (local) effects was recorded.
- Systemic toxicity:
Mortality/Viability: Twice daily.
Body Weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
- Ear thickness measurements: 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.
- Erythema scores:
Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system.
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth): 3
Severe erythema (beet redeness) to eschar formation preventing grading of erythema: 4

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD TG 429 Skin Sensitisation Local Lymph Node Assay
- Criteria used to consider a positive response: I DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures, including all amendments.
Classification of results
UN-GHS 2015; EC-CLP 2008 EC Hazard statement
SI < 3 No sensitizer
SI ≥ 3 Cat 1 Skin sensitizer H317: May cause an allergic skin reaction
EC3 value ≤ 2%: sub-category 1A
EC3 value > 2%: sub-category 1B

Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3)

TREATMENT PREPARATION AND ADMINISTRATION:
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.
-Induction: days 1,2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, 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 item.
-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 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) 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 then 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. Radioactivity 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: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
-Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
-Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. In addition a description of all other (local) effects was recorded.
Grading Irritation Reactions:
Erythema and eschar formation:
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth): 3
Severe erythema (beet redeness) to eschar formation preventing grading of erythema: 4
-Necropsy: No necropsy for gross macroscopic examination was performed according to study plan.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The results of a reliability test with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures are summarized in APPENDIX 2 of the study report. For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed at half year intervals during at least the past 9 years showing reproducible and consistent positive results.
The SI values calculated for the item concentrations 5, 10 and 25% were 1.4, 1.5 and 4.3 respectively. An EC3 value of 18.0% 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 16.5, 14.5, 13.4, 14.1, 17.3 and 9.8%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
50%
Cellular proliferation data / Observations:
MAIN TEST
CELLULAR PROLIFERATION DATA
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 for any of the animals.

DETAILS ON STIMULATION INDEX CALCULATION
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 1019, 1496 and 1329 DPM, respectively. The mean DPM/animal value for the vehicle control group was 943 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.1, 1.6 and 1.4 respectively.

EC3 CALCULATION
Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3) (Reference: Basketter DA, Lea LJ, Dickens A, Briggs, D, Pate I, Dearman RJ and Kimber I. A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl Toxicol 1999;19:261-266.)

CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.
No erythema was observed in any of the animals.
White test item remnants were present on the dorsal surface of the ears of animals throughout all dosing groups (between Days 1 and 5), which did not hamper scoring of the skin reactions.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Pre-screen Test

No erythema or signs of systemic toxicity were observed in any of the pre-screen animals.

White test item remnants were present on the dorsal surface of the ears of all animals (between Days 1 and 4), which did not hamper scoring of the skin reactions.

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 item concentration selected for the main study was a 50% concentration.

 

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the study results, Butanedioic acid, sulfo-, 4-C16-18 (even numbered)-alkyl esters, disodium salts would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item 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 (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labeling and packaging of items and mixtures (including all amendments).
Executive summary:

Assessment of skin sensitization was done with read-across substance Butanedioic acid, sulfo-, 4-C16-18 (even numbered)-alkyl esters, disodium salts in the Mouse (Local Lymph Node Assay). 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 item 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 item 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 the vehicle alone (Propylene glycol).

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 erythema was observed in any of the animals. 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 for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 1019, 1496 and 1329 DPM, respectively. The mean DPM/animal value for the vehicle control group was 943 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.1, 1.6 and 1.4, respectively.

Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 50%, Butanedioic acid, sulfo-, 4-C16-18 (even numbered)-alkyl esters, disodium salts was not considered to be a skin sensitizer.

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

Based on these results, Registered substance would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A guinea pig maximisation test was conducted with registered substance according to OECD/EC guidelines and GLP principles (WIL Research Europe, 2015). Based on a preliminary irritation study, the test substance concentrations selected for the main study were a 0.5% concentration for the intradermal induction and a 10% concentration for the epidermal induction exposure. A 10% test substance concentration was selected for the challenge phase. During the main study, no mortality occurred and no symptoms of systemic toxicity were observed. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Epidermal exposure to the vehicle or to 10% test substance did not result in a skin reaction in any of the animals.

A modified Draize-Shelanski repeat insult patch test was performed with human volunteers. Since the purity and amount of the test substance is unclear (text mentions 300 mg, whereas the title suggests 2.5% for induction, 1% challenge in petrolatum), and the vehicle is not mentioned, no conclusions can be drawn based on the outcome and the study was disregarded.

A mouse Local Lymph Node (LLNA) assay was done with read-across substance butanedioic acid, sulfo-, 4-C16-18 (even numbered)-alkyl esters disodium (CAS 90268 -39 -6) (Charles River, 2016).Test item concentrations were10, 25 or 50% w/w.No erythema was observed in any of the animals.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 for any of the animals.The SI values calculated for the test item concentrations 10, 25 and 50% were 1.1, 1.6 and 1.4, respectively.Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 50%, it was not sensitizing.

A supporting modified Draize-Shelanski repeat insult patch test with 50% read-across substance disodium isodecyl sulfosuccinate (CAS 37294 -49 -8) was applied to skin of human volunteers. The test report does not mention skin effects in the volunteers, however no conclusions could be drawn (Kligman, 1976).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the absence of sensitisation in a guinea pig maximisation test and negative LLNA study with read-across substance, the registered substance is found to be non-sensitising. The substance is thus not classified for sensitising properties according to CLP Regulation (EC) No. 1272/2008.