1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-, ethyl ester

Endpoint summary

Description of key information

One key study completed under GLP conditions and in accordance with OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay) and EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Sept. to 9th Oct. 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other:

Sex:

female

Details on test animals and environmental conditions:

At the start of the study the mice were in the weight range of 18.9 to 22.9 grams and were eight to twelve weeks old. Animals were acclimatised for 7 days prior to the study. Free access to mains drinking water and food was allowed through out the study. The temperature and relative humidity were set to achieve limits of 21+- 2 C and 40 to 70% respectively. The lighting was controlled by a time switch to give 12 hours continuous light and 12 hours of darkness.

Vehicle:

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

Concentration:

test material at concentrations of 5%, 10% or 25% w/w

No. of animals per dose:

Three groups of five animals each were treated at each concentration group and a further group of five animals were treated as a control group with acetone/olive oil alone.

Details on study design:

The mice were treated by daily application of 25 μl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3) by using a micropipette whose tip is used to spread the formulation over the dorsal surface of each ear. On Day 6 all mice were injected with 250 ul of a phophate buffered saline solution containing 3H-methyl thymidine (3HTdR ) to the tail vein giving a total of 20 uCi to each mouse. All animals were observed twice daily on Day 1, 2 and 3 and once daily on days 4 ,5, and 6. Any signs of toxicity or ill health were noted. Body weights of each mouse were recorded before dosing and before termination. Five hours after administration of 3HTdR all mice were killed by carbon dioxide asphyxiation and their auricular lymph nodes were excised and a 1 ml of phosphate buffered saline was added to each set of lymph nodes. Preparation of a single cell suspension was completed for the lymph nodes cells for each individual animal following standard procedures. Determination of the 3HTdR incorporation was completed by measuring radioactive disintegration for each animal's lymph node cells by using a beta-scintillation counter.

Positive control substance(s):

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

Statistics:

Analysis of variance (ANOVA) was carried out on the data followed by Barlett's test for analysis of homogenicity of variance. Comparisons were made beween the control and treatment groups using Dunnett's test.

Positive control results:

α-Hexylcinnamaldehyde, was considered to be a sensitiser under the conditions of the test with test control ration obtained for 25% v/v HCA at 8.4.

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

at 5%

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

at 10%

Key result

Parameter:

SI

Value:

0.6

Test group / Remarks:

at 25%

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Greasy fur was noted for all control and test animals. Particles on the ears was noted post dose from day 1 and resolved by day 5.

Concentration (% w/w) in acetone   Stimulation Index (SI)  Results  5  0.7  negative  10  0.8  negative  25  0.6  negative

Interpretation of results:

not sensitising

Conclusions:

The test material was considered to be a non-sensitiser under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

A stimulation index of less than 3 was recorded for the three concentrations of the test material and substance is considered not a skin sensitizer according to EU CLP regulations.