Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-04-13 to 14-05-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline and GLP compliant study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report):Sodium Erythorbate
- Substance type:White crystalline powder
- Physical state: Suspension in propylene glycol.
- Analytical purity: 99.62%
- Purity test date: 28 March 2013
- Lot/batch No.:201303731
- Expiration date of the lot/batch:27 March 2015
- Storage condition of test material:room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 15-23g
- Housing: Suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: 2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK ad libitum
- Water: Mains tap water ad libitum
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70%,
- Air changes (per hr): fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
Main test: 25%, 10% or 5% (w/w) in propylene glycol
No. of animals per dose:
Four mice per dose
Details on study design:
- Compound solubility: the test item was freshly prepared as a suspension in propylene glycol. This vehicle was chosen as it produced the highest suitable formulation at the required concentration (Appendix 3).

Preliminary screening test: Using available information regarding the irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item at 25% w/w in propylene glycol to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale in Table A below. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

No signs of systemic toxicity (Table 1), visual local skin irritation (Table 2) or irritation indicated by an equal to or greater than 25% increase in mean ear thickness (Table 3) were noted. Based on this information the test item at concentrations of 25%, 10% or 5% w/w in propylene glycol was selected for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The reliability of this test has been assessed within a six month interval of the study reported here. Positive control study used for comparison: 03 January 2013 to 09 January 2013 (Study number: 041206039; Appendix 1). Phenylacetaldehyde (>90%) was used as the positive control. A group of five animals was treated with 50 μl (25 μl per ear) of Phenylacetaldehyde (>90%) as a solution in propylene glycol at a concentration of 2.5% v/v. A further control group of five animals was treated with propylene glycol alone. In 2.5% (v/v) propylene glycol, the stimulation index of Phenylacetaldehyde was 6.32 and the result was positive. Phenylacetaldehyde (>90%) was considered to be a sensitizer under the conditions of the test.

TREATMENT PREPARATION AND ADMINISTRATION: The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse. Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The cell suspensions were then washed twice with PBS and precipitated with 5% trichloroacetic acid (TCA) at 2-8C overnight. Pellets were recovered by centrifugation and re-suspended in 1 mL TCA. 3HTdR incorporation was measured by β-scintillation counting (Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).)

Results and discussion

Positive control results:
Positive control study used for comparison: There was no positive control study conducted concurrently. However, the reliability of the test system was confirmed by the most recent positive control assay (Phenylacetaldehyde (>90%) ; 03 January 2013 to 09 January 2013; Study number:041206039; Appendix 1).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer." The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are indicated in Table B below.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 4.

Any other information on results incl. tables

Table B: Estimation of the Proliferative Response of Lymph Node Cells

Concentration (% w/w) in propylene glycol Stimulation Index Result
5 1.13 Negative
10 0.91 Negative
25 1.29 Negative

Study report attachments:

Table 1 Clinical Observations, Body Weight and Mortality Data – Preliminary Screening Test

Table 2 Local Skin Irritation – Preliminary Screening Test

Table 3 Measurement of Ear Thickness and Mean Ear Thickness Changes –Preliminary Screening Test

Table 4 Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Table 5 Individual Clinical Observations and Mortality Data

Table 6 Individual Body Weights and Body Weight Change

Appendix 1 Current Positive Control Study for the Local Lymph Node Assay

Appendix 3 Vehicle Determination Record

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
2,3-didehydro-3-O-sodio-D-erythro-hexono-1,4-lactone was considered to be a non-sensitizer under the conditions of the test.
Executive summary:

In a dermal sensitization study (41301073) with 2,3-didehydro-3-O-sodio-D-erythro-hexono-1,4-lactone (5, 10, 25% w/w in propylene glycol), young adult female CBA/Ca (CBA/CaOlaHsd) mice (4/group) were tested using the local lymph node assay (LLNA). The reliability of the test system was confirmed by the most recent positive control assay (Phenylacetaldehyde (>90%) in propylene glycol; January 2013).

There was no mortality and all animals appeared normal throughout the study. There were no statistically significant differences observed between any treatment groups with respect to body weight. Treatment with 2,3-didehydro-3-O-sodio-D-erythro-hexono-1,4-lactone at 5, 10 or 25% (w/w) resulted in stimulation indices of 1.13, 0.91 and 1.29 respectively.

In this study, 2,3-didehydro-3-O-sodio-D-erythro-hexono-1,4-lactone is not a potential skin sensitizer.