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In vitro genotoxicity assays:

I/In a GLP bacterial reverse assay (Sire 2011) perfomed following the OECD 471 guideline, the selected treatment-levels were ranged from 156.3 to 5000 µg/plate.

 A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at all tested dose-levels.

In the absence of S9 mix, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at 5000 µg/plate in the TA 1535, TA 1537 and TA 98 strains and at dose-levels = 2500 µg/plate in the TA 100 and TA 102 strains.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strain.


In the presence of S9 mix, a moderate toxicity was noted at 5000 µg/plate in the TA 102, TA 98 and TA 1535 strains and at dose-levels = 3750 µg/plate in the TA 1537 and TA 100 strains.

No noteworthy increase in the number of revertant colonies was noted in the first experiment (using the direct plate incorporation method).

 Noteworthy increases in the number of revertants, exceeding the positive threshold, were noted in the TA 1537 and TA 98 strains in the second experiment using the pre-incubation method (up to 3.3- and 2.8-fold the vehicle control value, respectively). These increases were then reproduced in the third experiment (up to 4.3- and 3.4-fold the vehicle control value, respectively). Consequently, they were considered as biologically relevant.

A slight increase in the number of revertants, remaining below the positive threshold (up to 1.9-fold the vehicle control value) was noted in the TA 100 strain in the second experiment. Then an increase in the number of revertants, exceeding the positive threshold was noted in the third experiment (up to 3.5-fold the vehicle control value), without any clear evidence of a dose-response relationship. Since the increase did not exceed the positive threshold in the second experiment and was not dose-related in the third experiment, it remained equivocal.

In Conclusion, the test item showed mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium,after metabolic activation system with S9 mix.

 

II/ In the vitro chromosome aberration test (Sire 2011) was performed according to the international guidelines (OECD No. 473 and Council Regulation No. 440/2008 Part B.10) and in compliance with the principles of Good Laboratory Practice.

The objective of this study was to evaluate the potential of the test item to induce chromosome aberrations in cultured human lymphocytes.

Experiments without S9 mix

Cytotoxicity

Following the 3-hour treatment, a slight to severe toxicity was noted at dose-levels >= 125 µg/mL, as shown by a 31-100% decrease in the MI.

Following the 20-hour treatment, a moderate to marked toxicity was induced at dose-levels >= 125 µg/mL, as shown by a 47-77% decrease in the MI.

Following the 44-hour treatment, a slight to moderate toxicity was induced at dose-levels >= 125 µg/mL, as shown by a 30-54% decrease in the MI.

 

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.  62.5, 125 and 250 µg/mL for the 3-hour treatment, the latter inducing a 43% decrease in the MI and higher dose-levels being too cytotoxic,

.   31.3, 62.5 and 125 µg/mL for the 20-hour treatment, the latter inducing a 47% decrease in the MI and higher dose-levels being too cytotoxic,

.    250 µg/mL for the 44-hour treatment, this dose inducing a 54% decrease in the MI.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted after the 3-, 20- as well as 44-hour treatments.

 

Experiments with S9 mix

Cytotoxicity

In the first experiment, a slight to severe toxicity was induced at dose-levels = 62.5 µg/mL as shown by a 30-100% decrease in the MI.

At the 20-hour harvest time in the second experiment, a moderate to marked toxicity was noted at dose-levels = 31.3 µg/mL, as shown by a 47-78% decrease in the MI.

At the 44-hour harvest time (second experiment), a moderate to marked toxicity was noted at dose-levels = 125 µg/mL, as shown by a 51-75% decrease in the MI.


Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.  31.3, 62.5 and 125 µg/mL for the 20-hour harvest time in the first experiment, the latter inducing a 50% decrease in the MI,

.  15.6, 31.3 and 62.5 µg/mL for the 20-hour harvest time in the second experiment, the latter inducing a 50% decrease in the MI,

.  125 µg/mL for the 44-hour harvest time, this dose inducing a 51% decrease in the MI.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times.

The test item did not induce chromosome aberrations in cultured human lymphocytes.

III/ In vitro mammalian cell gene mutation assay (Sire 2011) was performed according to OECD 476, and according to GLP:

In the absence of S9, the selected dose-levels were as follows:

. 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL for the first experiment (3-hour treatment),

. 3.13, 6.25, 12.5, 25, 50, 75 and 100 µg/mL for the second experiment (24-hour treatment).

 

At the end of the treatment periods, a precipitate was observed in the culture medium at dose-levels = 75 µg/mL.

Following the 3-hour treatment, a moderate toxicity was induced at 100 µg/mL, as shown by a 57% decrease in the Adj. RTG.

Following the 24-hour treatment, a marked to severe toxicity was induced at dose-levels = 50 µg/mL, as shown by a 74-98% decrease in the Adj. RTG.

Following the 3-hour treatment, no noteworthy increase in the mutation frequency was noted up to the dose-level of 100 µg/mL, inducing 57% decrease in the Adj. RTG and showing precipitate in the culture medium.

Following the 24-hour treatment, no increase in the mutation frequency which could be considered as biologically relevant was noted up to the highest selected dose-level of 100 µg/mL, showing precipitate in the culture medium and inducing a 98% decrease in the Adj. RTG.

 

In the presence of S9, the selected dose-levels were 6.25, 12.5, 25, 50, 100 and 200 µg/mL for both experiments (3-hour treatments).

At the end of the treatment, a precipitate was observed in the culture medium at dose-levels = 100 µg/mL.

 

In the first experiment, a slight to moderate toxicity was induced at dose-levels = 100 µg/mL, as shown by a 37-66% decrease in the Adj. RTG.

In the second experiment, a moderate toxicity was induced at 200 µg/mL, as shown by a 58% decrease in the Adj. RTG.

No noteworthy increase in the mutation frequency was observed in either experiment, up to the dose-level of 200 µg/mL, inducing up to 66% decrease in the Adj. RTG and showing precipitate in the culture medium.

The test item did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.

In vivo genotoxic assays:

The potential clastogenic activity of Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues

was tested in vivo in a GLP study using both the micronucleus test in bone marrow in the rat combined with the comet assay in the liver and the colon (Simar, 2012).

The potential clastogenic activity was tested using the in vivo micronucleus test in female and male rats, in compliance with the Commission Regulation (EC) No. 440/2008 and the OECD Guideline 474, by oral route, using 3 successive daily treatments (see ICH S2(R1) and scientific developments and experience of the regulation of chemical compounds, a working group of the GUM), at the maximum dose recommended by OECD guidelines, i.e. 2000 mg/kg/day (x3), followed by one sampling time 3 to 6 hours after the last treatment. The two lower doses of 1000 and 500 mg/kg/day (x3) were also analysed. No statistically significant decrease in the ratio PCE to NCE was noted in the 3 test treatment groups when compared to the negative control group. No statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes was found in the animals treated with Dodecane-12-Lactam Manufacturing of, by-products from, distillation residue at any dose, both sexes combined or males and females separately, when compared with the control group.

The comet assay was investigated in the same study under alkaline conditions (SCGE) in the liver and the colon, in male and/or female OFA Sprague- Dawley rats. Animals were treated orally once a day for 3 consecutive days, 24 hours apart, at dose levels of 2000, 1000 and 500 mg/kg. The validity criteria for the results were fulfilled. The study was thus considered as valid. the test item did not present any DNA strand breaks and/or alkali-labile sites inducer activities toward hepatocytes or colon cells from OFA Sprague-Dawley male rats. As a conclusion, the test item induced no genotoxic activity under these experimental conditions.

Under the experimental conditions of this combined in vivo micronucleus test with the comet assay in the rat , the test item did not present any DNA strand breaks and/or alkali-labile sites inducer activities toward hepatocytes from OFA Sprague-Dawley male rats. Furthermore, in male and female OFA Sprague-Dawley rats, the test item induced no genotoxic activity in bone marrow cells. As a conclusion, the test item induced no genotoxic activity under these experimental conditions.




Justification for selection of genetic toxicity endpoint
The test item Dodecane-12-Lactam Manufacturing of, by-products from, distillation residue (batch April 2011), provided by ARKEMA, was investigated for genotoxic potential by the means of the in vivo micronucleus test in bone marrow and the in vivo comet assay under alkaline conditions (SCGE) in the liver and the colon, in male and/or female OFA Sprague- Dawley rats. Animals were treated orally once a day for 3 consecutive days, 24 hours apart, at dose levels of 2000, 1000 and 500 mg/kg.
The validity criteria for the results were fulfilled. The study was thus considered as valid.

Short description of key information:
The substance which induced mutagenic activity in the ames test in presence of metabolic activation in two strains (TA1537 and TA 98) did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after three oral administrations at the dose-levels of 500, 1000 and 2000 mg/kg/day and did not induce DNA damage in hepatocytes or colon cells from SD male rats, treated once orally with 2000 and 1000 mg/kg at the same exposure. On a weight of evidence basis, the substance is considered as not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification is warranted for germ cells mutagenicity under EU Dangerous Substances Directive 67/548/EEC or CLP EU Regulation 1272/2008.

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