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Genotoxicity in vitro:

- Bacterial reverse mutation assay: one study (Scarcella, RTC report, 1999) was chosen as key study,

of reliability 1 and one other study (Haworth et al. 1983) of reliability 2 was chosen as supporting study.

The first is very well described and followed the current guidelines with GLP, the second is equivalent

to the guidelines and well described too.

In the RTC report of 1999, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 were used.

Two experiments were performed, one using a plate incorporation method, the other using a pre incubation method.

In both experiments the test substance was assayed at the dose levels of 5000, 2500, 1250, 625 and 313 µg/plate.

In the plate incorporation assay slight toxicity was observed with tester strain TA1537 both in the absence or presence

of S9 metabolism at the highest dose level. In the pre incubation assay, with all tester strains, severe toxicity,

as indicated by microcolony formation, was observed at the highest dose level in the absence of S9 metabolism.

The test substance did not induce two-fold increases in the number of revertant colonies at any dose level,

in any tester strain, in the plate incorporation or pre incubation assay, in the absence or presence of S9 metabolism.

In the publication of Haworth et al. 1983, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were used,

by 2 different laboratories with a modified preincubation method. The test concentrations were: 0, 3.3, 10, 33,

100 and 167 µg/plate in one laboratory and 0, 100, 333, 1000, 3333 and 5000 µg/plate in another. In the absence

and in the presence of metabolic activation system, PMP does not induce any reverse mutation in all strains tested.

Based on these 2 studies, PMP does not induce reverse mutation in Salmonella typhimurium tested strains.

- mammalian cell gene mutation: one study (Cinelli, RTC report, 1999) was chosen as key study, of reliability 1.

It is a GLP study, which followed the current guidelines. It used Chinese hamster lung fibroblasts (V79) with target

gene HGPRT. In this assay for 6-thioguanine resistance, V79 cells cultured in vitro were exposed to

paramethoxyphenol ecaille (DMSO as solvent), at concentrations of:

- 2500, 1250, 625, 313, 156 and 78.1 µg/mL, in the absence of S9 mix,

- and 3750, 2500, 1250, 625, 313 and 156 µg/mL in the presence of S9 mix, for the first experiment and

- 2500, 1900, 1250, 625, 313 and 156 µg/mL for the second experiment.

Paramethoxyphenol ecaille was tested up to 2500 and 3750 µg/mL in the first mutation assay in the absence

and presence of S9 metabolism, respectively.

Based on this study, PMP does not induce mutation in V79 cells, either in the absence or presence

of S9 metabolic activation.

An other study is available for mammalian cell gene mutation (Rogers-Back A, 1986), but it is of reliability 3

because the test substance is not clearly defined and the effects were observed at highly toxic doses.

In this study on mouse lymphoma L5178Y cells, the test substance produced a positive response in the

absence of exogenous metabolic activation.

- mammalian chromosome aberration: one study (Ciliutti, RTC report, 1999) was chosen as key study,

of reliability 1. It is a GLP study, which followed the current guidelines. It was carried out on primary human

lymphocyte cultures, which were exposed to Paramethoxyphenol ecaille, prepared in DMSO, at concentrations of:

- 5000, 2500, 1250, 625, 313, 156, 78.1, 39.1 µg/mL (experiment 1), with and without metabolic activation

- 156, 78.1, 39.1, 19.5, 9.77, 4.88, 2.44, 1.22, 0.611 µg/mL (experiment 2), without metabolic activation.

Paramethoxyphenol ecaille was tested up to cytotoxic concentration: 5000 µg/ml.

Based on this study, PMP does not induce chromosomal aberration in cultured human lymphocytes,

either in the absence or presence of S9 metabolic activation.

Seven other studies are available but the reliability is 3.

Three Ames test and one sister chromatide exchange assay (in vitro) were performed on PMP and the results

are negative.

One DNA synthesis and repair assay showed that PMP induce a decrease of DNA synthesis and repair but this

result was not PMP specific and the data was too poor to permit conclusions.

The two last studies of reliability 3 are one mammalian cell gene mutation and one mammalian chromosome

aberration tests, but the test substance was not clearly defined in the reports.

Genotoxicity in vivo:

Only one study (Esber, 1986) was available in this endpoint, it is an in vivo mammalian bone marrow chromosome

aberration assay, which led to a negative result. But this study is of reliability 3 because the test material is not

clearly identified and all the test conditions and details were not available to conduct an assessment.

Conclusion for genotoxic potential of PMP: Based on three in vitro studies performed on our substance

(Scarcella , Cinelli and Ciliutti, RTC reports, 1999), PMP ecaille was considered to be not genotoxic.


Justification for selection of genetic toxicity endpoint
Several studies were selected for assessment.

Short description of key information:
Several in vitro tests performed on PMP are available. All results coming from reliable studies are negative. Only one in vivo test has been generated
on PMP. The result is also negative; however the reliability of this study is 3 according to Klimisch rating.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on several reliable in vitro studies performed on our substance, PMP was considered to be not genotoxic.