(6R-trans)-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 January 2010 - 01 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and EC guidelines and according to GLP principles.

Justification for data waiving:

other:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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:

(6R-trans)-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

EC Number:

244-870-1

EC Name:

(6R-trans)-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Cas Number:

22252-43-3

Molecular formula:

C8H10N2O3S

IUPAC Name:

7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Constituent 2

Reference substance name:

7-ADCA

IUPAC Name:

7-ADCA

Details on test material:

- Physical state: White powder
- Analytical purity: 99.0%
- Lot/batch No.: D212013
- Expiration date of the lot/batch: 28 February 2011 (retest date)
- Stability under test conditions: Stable
- Storage condition of test material: In refrigerator (2-8°C) in the dark
- Other: pH is 3.6 at concentration of 0.1%; Not stable at higher temperatures.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: Young adult animals (approx. 10 weeks old) were selected.
- Weight at study nitiation: 18-21 gram
- Housing: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment.
- Diet: ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: ad libitu, tap water
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).

ENVIRONMENTAL CONDITIONS
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 19.3 – 23.5ºC), a relative humidity of 40-70% (actual range: 17 - 80%) and 12 hours artificial fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: From: 11 January 2010 To: 01 February 2010

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

10%, 25%, 50%

No. of animals per dose:

5

Details on study design:

Main study: three experimental groups of five female CBA/J mice were treated with test substance 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 vehicle alone (Acetone/Olive oil (4:1 v/v)).
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. 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.

Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.
Preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.

A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. Two test substance concentrations were tested; a 50% and 25% concentration. The highest concentration was the maximum concentration as required in the test guidelines.

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by NOTOX. In this study, performed in September/October 2009, females of the CBA/J mouse strain were checked for sensitivity to Alpha- Hexylcinnamaldehyde, technical grade. The females were approx. 11 weeks old at commencement of the study. HCA concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 (v/v)).

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: test substance SI ± SEM 0% (vehicle) 1.0 ± 0.3 25% 1.3 ± 0.3 50% 1.1 ± 0.2 100% 1.3 ± 0.4

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: test substance mean DPM ± SEM 0% (vehicle) 339 ± 68 25% 426 ± 58 50% 360 ± 29 100% 451 ± 102

Any other information on results incl. tables

The slight irritation of the ears as shown by two animals treated at 25% and all animals treated at 50% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined.

The majority of auricular lymph nodes were considered normal in size, except for one enlarged node of one animal treated at 50% and one enlarged node in one control animal. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

The DPM values from the control animal and experimental animal (50% treated) with the unilateral enlarged nodes were outliers and considered to be background findings for this batch of animals and therefore not used for interpretation. The DPM value for the control animal was also outside the range expected for vehicle treated animals.

Reliability check:

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.4, 1.2 and 5.1 respectively. An EC3 value of 16.9% was calculated using linear interpolation. 

The calculated EC3 value was found to be in the acceptable range of 2 and 20%. Theresults of the 6 monthlyreliability checks of the recent years were13.1, 15.6, 14.1, 13.8, 13.9, 16.0 and 11.9%. 

Based on the results, it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Since there was no indication that the test substance elicits an SI = 3 when tested up to 50%, 7-ADCA was considered not to be a skin sensitizer. It was established that the EC3 value (if any) exceeds 50%.