Reaction mass of ethyl (1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate and ethyl (1R,2R,4R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 April 2010 to 18 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted guidelines and to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

TM 09-218

IUPAC Name:

TM 09-218

Test material form:

other: Clear liquid

Details on test material:

- Name of test material (as cited in study report): TM 09-218
- Structural formula attached as image file (if other than submission substance): see Fig.
- Physical state: Clear liquid
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd.
- Age at study initiation: eight to twelve weeks
- Weight at study initiation: 16.9 to 20.9 g
- Housing: Individually in polycarbonate cages with woodflake bedding
- Diet (e.g. ad libitum): Rat and Mouse No. 1 Maintenance Diet ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40 to 70
- Air changes (per hr): The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 06 April 2010 To: 20 April 2010

Animal housing, diet and water supply
Animals were housed inside a barriered rodent facility (Building F21, Room 061/062). The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms.

The mice were allocated without conscious bias to cages within the treatment groups. They were housed individually in polycarbonate cages with woodflake bedding. The mice were also given Nestlets and a plastic shelter for environmental enrichment. Certificates of analysis for woodflake bedding and Nestlets were lodged in Huntingdon Life Sciences Ltd. Archives.

The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature and relative humidity controls were set to maintain the range of 19 to 23°C and 40 to 70% respectively. Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study. Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.

Periodic checks were made on the number of air changes in the animal rooms. Temperature and humidity were monitored daily and records are archived with the departmental raw data.

Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available to be automatically brought into operation should the public supply fail.

The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.

Each batch of diet was analysed routinely by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinised and approved before any batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis were received routinely from the water supplier. Since the results of these various analyses did not provide evidence of contamination that might have prejudiced the study, they are not presented.

No other specific contaminants that were likely to have been present in the diet or water were analysed, as none that may have interfered with or prejudiced the outcome of the study was known.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25% v/v, 50% v/v & as supplied

No. of animals per dose:

4

Details on study design:

Test substance preparation
A vehicle trial was conducted and TM 09-218 formed a clear solution at a concentration of 50% v/v in acetone:olive oil (4:1 v/v). It was also considered suitable to dose ‘as supplied’.

Formulation
The dose level for the preliminary investigation was chosen based on the physical properties of the test substance e.g. solubility, viscosity. The doses for the main phase of the study were based on the results of the preliminary investigation.
Determination of the homogeneity, stability and purity of the test substance or test substance formulations were not undertaken as part of this study.
Detailed records of test substance usage were maintained. The amount of test substance necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.
The test substance was used as supplied.
The test substance formulations were prepared on the day of dosing at the required concentrations.
The absorption of the test substance was not determined.

Selection of dose levels
The maximum practical concentration for pinna dosing was ‘as supplied’. Based on this information this concentration was selected for the preliminary investigation. The results of the preliminary investigation indicated that ‘as supplied’ was a suitable high dose level for the main phase of the study. Based on this information the following concentrations were selected for the main phase of the study:
25 and 50% v/v and as supplied
Prior to dose administration the Study Director authorised the choice of vehicle and dose levels in the raw data.

Administration of test substance
Preliminary investigation
One female was treated with the test substance ‘as supplied’. The mouse was treated by daily application of 25 μl of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of the ear using the tip of the pipette.

Main phase
Groups of four mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 μl of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.

Administration of 3H-methyl Thymidine
In the main phase of the study, five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered saline containing 3H-methyl Thymidinea (3HTdR: 80 μCi/mL) giving a nominal 20 μCi to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle after the mouse had been heated in a warming chamber.

Observations
Clinical signs
All animals were observed daily for signs of ill health or toxicity. The ears were also examined for signs of irritation.

Bodyweight
The weight of the mouse in the preliminary investigation was recorded on arrival, Day 1 (first day of dosing) and prior to termination (Day 4). These data are not reported. The weight of each mouse in the main phase of the study was recorded on arrival (these data are not reported), Day 1 (first day of dosing) and prior to termination (Day 6).

Terminal studies
Termination
The mouse in the preliminary investigation was humanely killed by carbon dioxide asphyxiation on Day 4 of the study. No further investigations were carried out with this animal.
In the main phase of the study, five hours following the administration of 3HTdR on Day 6 all mice were humanely killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised and pooled for each experimental group. 1.0 mL of phosphate buffered saline was added to the pooled lymph nodes for each group. The animals were then discarded and no further investigations were carried out. The following procedures were carried out with the lymph nodes from these animals only.

Positive control substance(s):

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

Results and discussion

Positive control results:

A study was performed to confirm the sensitivity and reliability of the experimental technique used at Huntingdon Life Sciences to detect skin sensitization potential. The study was performed using the murine local lymph node assay (OECD 429 individual animal approach) and a known moderate sensitizer – hexyl cinnamic aldehyde (HCA). This study was conducted between the 14 and 20 October 2009 using ten mice of the CBA/Ca strain supplied by Harlan UK Ltd., Bicester, Oxon, England. The HCA was supplied by Sigma Aldrich Chemical Co. England (Lot No. 02002DH, expiry 17 October 2009). The mice were treated by daily application of 25 μL of HCA, or vehicle control to the dorsal surface of both ears for three consecutive days. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio). The positive control study is considered to be valid if the results from the HCA group have a three-fold greater increase in 3HTdR incorporation compared to control values.
In this assay the test/control ratio obtained for HCA at 25% was 6.0. This indicates that HCA demonstrates the potential to induce skin sensitization(delayed contact hypersensitivity) and confirms the sensitivity of the technique to detect sensitization potential.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The EC3 is the concentration of test substance which will result in a stimulation index (SI) of 3. As one concentration has an SI greater than 3 and one concentration has an SI of less than 3 then the EC3 was calculated using the formula: EC3 = c+[(3-d)/(b-d)](a-c) a = concentration giving SI greater than 3, b – SI at concentration a, c = concentration giving SI less than 3, d = SI at concentration c. The EC3 value is 50% v/v.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: From table one below it can be seen that the dpm increase with increasing dose. The test/control ratios are >3 for the 50% and as supplied doses, giving positive results indicating potential for sensitisation.

Any other information on results incl. tables

Preliminary investigation

Mortality and clinical signs

There were no deaths and no signs of ill health or toxicity observed during this study. However, wet fur on the cranial region was noted post-dose from Day 1, resolving completely by Day 4.

No signs of irritation were seen over the dosed area during the study.

 

Bodyweight

The animal gained weight during the study. On the basis of the results from the preliminary investigation, ‘as supplied’ was considered a suitable high dose level for the main phase of the study.

 

Main phase

Mortality and clinical signs

There were no deaths and no signs of ill health or toxicity observed during this study. However the following observations were noted:

Vehicle control - greasy fur on the cranial region was seen post-dose in all animals from Day 1, prior to dosing for all animals on Day 3 and in one animal on Day 4. The sign had resolved completely for all animals by Day 5.

25% v/v - greasy fur on the cranial region was seen post-dose in all animals from Day 1, prior to dosing for all animals on Day 3, in all animals on Day 4 and in three animals on Day 5.

The sign had resolved completely in all animals by Day 6.

50% v/v - greasy fur on the cranial region was seen post-dose in all animals from Day 1, prior to dosing for all animals on Day 3 and in three animals on Day 4. The sign had resolved completely in all animals by Day 5.

‘As supplied’ – greasy fur on the cranial region was seen post-dose in all animals from Day 1.

The sign had resolved completely in all animals by Day 4. No signs of irritation were seen over the dosed area during the study.

 

Bodyweight

Aloss in bodyweight was recorded for one female (No. H3) in Group 2 and one female (No. H8) in Group 3 during the study. All remaining animals gained weight during the study.

Interpretation of results

The test substance is regarded as a sensitizer if at least one concentration of the test substance results in a three-fold greater increase in 3HTdR incorporation compared to control values. The EC3 is the concentration of test substance which will result in a stimulation index (SI) of 3. As one concentration has an SI greater than 3 and one concentration has an SI of less than 3 then the EC3 was calculated using the formula:

EC3 = c+[(3-d)/(b-d)](a-c)

a = concentration giving SI greater than 3, b – SI at concentration a, c = concentration giving SI less than 3, d = SI at concentration c.

Table 1 Group dpm/node and test/control ratios

Group	Concentration% v/v	dpm	Number of lymph nodes per group	dpm/node	Test/control ratio†	Result+ = positive- = negative
2	AOO	5714.70	8.0	714.34	n/a	n/a
3	25	7544.70	8.0	943.09	1.3	-
4	50	16891.40	8.0	2111.43	3.0	+
5	As supplied	20440.00	8.0	2555.00	3.6	+

† Test/control ratio of 3 or greater indicates a positive result

n/a Not applicable

dpm Disintegrations per minute (less background count of 44.90 dpm)

AOO Acetone:olive oil (4:1 v/v) (vehicle control)

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

TM 09-218 is regarded as a potential skin sensitizer and the EC3 value is 50% v/v.

Executive summary:

The skin sensitisation potential was assessed for the test substance, TM 09-218, according to the OECD Test guideline 429. TM 09-218 was determined to be a potential skin sensitiser based on an EC3 value of < 25 % v/v.