(3R,4S,5R)-3,4,5-trihydroxycyclohex-1-enecarboxylic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

EC number

IUPAC Name:

EC number

Details on test material:

The test item was supplied by or on behalf of the Sponsor including the following information.
Identification: (-)-Shikimic acid
CAS No. 138-59-0
Roche No. Ro0642565-000
Batch: 1329R037/ Lot. No. 0700791872
Purity: min. 98.0%
Expiry Date: 22 August 2014
Storage Conditions: At room temperature, protected from light*
Stability in Solvent: Not indicated by the sponsor
* only valid for storage, not for test performance

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS: mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: 18.1 - 23.3 g
- Housing: group caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

The test item in the main study was assayed at 5, 10 and 25%.

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used, was a 25% solution in DMSO. Vortexing of the test item was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6 (for detailed results see Appendix 1).
At the tested concentrations the animals did not show any signs of systemic toxicity. Between day 3 and day 6, both animals treated with 10 or 25% test item concentration, respectively, showed an erythema of the ear skin (Score 1).
Thus, the test item in the main study was assayed at 5, 10 and 25%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10 and 25% in DMSO. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (  8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE

Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). Approximately five hours after treatment with 3HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetíc acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ?-scintillation counter.

INTERPRETATION OF RAW DATA

The proliferation response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiliser in the LLNA if the following criteria are fulfilled:
-First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
-Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

OBSERVATIONS

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.

Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.

Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).

Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.

Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation).
The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2007). No outlier animal was detected in the Dean-Dixon-Test.

Results and discussion

Positive control results:

Experiment performed in April 2013 (Harlan study number 1541000) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 1.6, 2.4, and 5.9, respectively.
The EC3 value calculated was 12.6 % (w/v).
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Results

Results 1

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see depicted table below

Results 2

Parameter:

SI

Value:

0.97

Test group / Remarks:

5% test item

Results 3

Parameter:

SI

Value:

0.66

Test group / Remarks:

10% test item

Results 4

Parameter:

SI

Value:

0.72

Test group / Remarks:

25% test item

Any other information on results incl. tables

Table 1: Calculation and Results of Individual Data

Vehicle: DMSO

Test item concentration			DPM values measured	DPM-BG per animal (2 lymph nodes)^a)	S.I.^b)
% (w/w)	Group no.	Animal no.
---	---	BG I	43	---	---
---	---	BG II	37	---	---
0	1	1	1310	1270.0	---
0	1	2	888	848.0	---
0	1	3	1608	1568.0	---
0	1	4	2110	2070.0	---
0	1	5	1618	1578.0	---
5	2	6	916	876.0	0.6
5	2	7	2867	2827.0	1.9
5	2	8	810	770.0	0.5
5	2	9	1549	1509.0	1.0
5	2	10	1208	1168.0	0.8
10	3	11	979	939.0	0.6
10	3	12	779	739.0	0.5
10	3	13	1377	1337.0	0.9
10	3	14	1373	1333.0	0.9
10	3	15	516	476.0	0.3
25	4	16	402	362.0	0.2
25	4	17	841	801.0	0.5
25	4	18	1377	1337.0	0.9
25	4	19	1763	1723.0	1.2
25	4	20	1113	1073.0	0.7

1    =  Control Group

2-4=  Test Group

^a)   =  values corrected for mean background value (BGI and BGII)

^b)    =  Stimulation Indices relative to the mean of the control group (Group 1)

Table 2: Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)^a)	SD	S.I.
Vehicle Control Group (DMSO)	1466.8	449.5	1.0
5% (-)-Shikimic acid	1430.0	832.0	0.97
10% (-)-Shikimic acid	964.8	375.7	0.66
25% (-)-Shikimic acid	1059.2	517.4	0.72

^a)      Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symtoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item (-)-Shikimic acid was not a skin sensitiser under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of (-)-Shikimic acid, three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10 and 25% (w/w) in DMSO by topical application to the dorsum of each ear for three consecutive days. A control group of five mice was treated with the vehicle (DMSO) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of³H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 0.97, 0.66 and 0.72 were determined with the test item at concentrations of 5, 10 and 25% in DMSO. A dose response was not observed.

The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.