(2S,3S,5R)-3-methyl-7-oxo-3-(1H-1,2,3-triazol-1-ylmethyl)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4,4-dioxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report Date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

Constituent

Details on test material:

- Appearance: white powder.
- Storage conditions: room temperature.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelman GmbH
- Age at first administration: 8 weeks
- Weight at first administration: 18 -22 g
- Housing: Single caging. Makrolon cages type II.
- Diet: Ssniff maintenance diet R/M-H (item V1534-3), ad libitum.
- Water: Tap water from Makrolon-bottles with staineless steel canules, ad libitum.
- Acclimation period: 6 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Average of 21.
- Humidity (%): Average of 53.6
- Photoperiod (hrs dark / hrs light): artificial light from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

100 %

Concentration:

0 - 10 - 25 - 33.1 % (w/w) test substance in dimethylformamide.

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 33.1% is the maximal achievable concentration
- Irritation: no excessive local skin irritation at the application sites or an important increase in ear thickness at 33.1 %.
- Lymph node proliferation response: not investigated.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay.
- Criteria used to consider a positive response: Results are presented as test/control ratio (Stimulation index SI). A substance is regarded as a sensitiser in the LLNA if the test substance induces a 3-fold or greater increase in 3H-methyl thymidine incorporation into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes, as indicated by the SI, together with the consideration of dose response.

TREATMENT PREPARATION AND ADMINISTRATION:
- The test substance solutions were prepared freshly on each day of administration by the solution of the test substance in DMF (w/w).
- A solution of 25 % HCA in acetone:olive oil was prepared freshly on every day of administration.
- The test substance was administered in 3 concentrations to the dorsal surfaces of the ears of the animals of the test substance groups. 25 µL were applied. In a manner identical to that of animals in the treatment groups animals of one negative control group and one positive control group were treated with DMF and HCA respectively. Each animal was treated for 3 consecutive days.

Positive control substance(s):

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

Statistics:

None, except for the calculation of means.

Results and discussion

Positive control results:

Application of 25 % HCA resulted in an SI of 29.9. This result proves the sensitivity of the strain of animals used and the reliability of the experimental technique.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

13.2

Test group / Remarks:

10 %

Results 2

Key result

Parameter:

SI

Value:

15.8

Test group / Remarks:

25 %

Results 3

Key result

Parameter:

SI

Value:

19.2

Test group / Remarks:

33.1%

Results 4

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

0 % (Negative control)

Results 5

Key result

Parameter:

SI

Value:

29.9

Test group / Remarks:

Positive control

Any other information on results incl. tables

- Mortality: All animals survived till the end of the study.

- General observations: No abnormal behaviour or clinical signs were detected during the experiment in the animals.

- Skin reactions: No local irritations were observed at the application sites of all animals of all test substance groups and the negative control group throughout the whole study.

- Body mass: Body masses and body mass gains of all animals were in the range to be expected from animals of the same strain, sex and age.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Conclusions:

Tazobactam Acid is regarded as a sensitiser in the LLNA, since the SIs of all examined test substance concentrations were clearly greater than 3.

Executive summary:

Method

The Local Lymph Node Assay was performed to evaluate a possible skin sensitising potential of Tazobactam Acid according to the OECD-Guideline 429 and the EC Directive 2004/73/EC, method B.42.

The test substance was solved in N,N-Dimethylformamide and was administered to three groups of 5 female CBA/Ca mice. Administration was performed epicutaneously to the dorsal surface of both ears, once a day on three consecutive days. The volume administered was 25 µL per ear. Concentrations used: 0 - 10 - 25 - 33.1 %.

Two groups with 5 animals each served as positive and negative controls.

5 days after the first topical application,³H-methyl thymidine was intravenously administered to all mice via a tail vein. Approximately 5 hours later all animals were sacrificed, the draining auricular lymph nodes were excised. The proliferation of the lymphocytes of the draining lymph nodes was measured by the determination of the amounts of incorporate³H-methyl thymidine into the cells.

The stimulation index (SI) was calculated as the ratio of the disintegrations per minute (dpm) of the dosed groups or of the positive control group to the dpm of the negative control group.

 

Results

All animals survived till the end of the study.

No adverse effects were noted in any animal.

Body masses and body mass gains were in the range to be expected from animals of the same strain, sex and age.

No skin irritating effects at the application sites were observed in all animals of the test substance groups and the negative control group throughout the whole study.

 

Stimulation indices

The calculated stimulation indices of the test substance concentrations were 13.2 (low dose), 15.8 (mid dose) and 19.2 (high dose). According to the guidelines the decision process with regard to a positive response includes a stimulation index of equal to or greater than 3, together with consideration of dose-response.

 

Positive control

The positive control substance led to a stimulation index of 29.9, thus demonstrating the validity of the experiment.

 

Conclusion

"Tazobactam Acid" is regarded as a sensitiser in the LLNA.