Amides of alkyldiamine, fatty acids and carboxylic acids

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 June 2007 to 08 August 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chrosome aberration test

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

impurity

Test material form:

solid: particulate/powder

Details on test material:

The test material is a UVCB substance.

Specific details on test material used for the study:

Identification AD-2000
Form White powder
Batch L-620150
Storage At room temp in the dark
Stability under storage condition Stable
Expiry date 1 January 2009

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other:

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes were used.
Blood was collected from healthy adult, non-smoker, male volunteers. The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2006) and are presented below:

Dose range finding study: age 30, AGT = 13.5 h First cytogenetic assay: age 34, AGT = 14.4 h Second cytogenetic assay: age 36, AGT = 13.9 h

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

With S9 mix: 3, 10 and 33 µg/ml culture medium
(3 h exposure time, 48 h fixation time).
Without S9 mix: 3, 10 and 100 µg/ml culture medium
(24 h exposure time, 24 h fixation time).
3, 10 and 100 µg/ml culture medium
(48 h exposure time, 48 h fixation time).

Justification
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test the test substance was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.

Lymphocytes (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of
the test substance for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.

The highest tested concentration was determined by the solubility of the test substance in the culture medium at the 3 h exposure time. At the 24 and 48 h exposure time, the test substance was tested beyond the limit of solubility to obtain adequate toxicity data.

After 3 h exposure to the test substance in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20-22 h (24 h fixation time). The cells that were exposed for 24 hand 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 hand 48 h fixation time).
Cytotoxicity of the test substance in the lymphocyte cultures was determined using the mitotic index.

Vehicle / solvent:

Dimethyl sulfoxide

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Cultured peripheral human lymphocytes were used as test system. Blood was collected from healthy adult, non-smoker, male volunteers. The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2006) and are presented below:

Dose range finding study: age 30, AGT = 13.5 h First cytogenetic assay: age 34, AGT = 14.4 h Second cytogenetic assay: age 36, AGT = 13.9 h

Evaluation criteria:

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range
b) The positive control substances should produce a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.

Statistics:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05)
increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number
of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided,
p < 0.05) increase in the number of cells with chromosome aberrations.

The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics

x^2=(N-1)(ad-bc)^2/((a+b)(c+d)(a+c)(b+d))


where b = the total number of aberrant cells in the control cultures.
d = the total number of non aberrant cells in the control cultures.
no = the total number of cells scored in the control cultures.
a = the total number of aberrant cells in treated cultures to be compared with the
control.
c = the total number of non aberrant cells in treated cultures to be compared with the
control
n1 = the total number of cells scored in the treated cultures
N = sum of n0 and n1



x^2>(N-1)(ad-bc)^2/((a+b)(c+d)(a+c)(b+d))


is small (p< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the
95% confidence level.

Results and discussion

Test results

Key result

Species / strain:

mammalian cell line, other: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Additional information on results:

Both in the absence and presence of S9-mix, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations
Both in the absence and presence of S9-mix, the test substance did not increase the number of polyploid cells and cells with endoreduplicated chromosomes

Applicant's summary and conclusion

Conclusions:

the test substance is not clastogenic in human lymphocytes under the experimental conditions described in this report.

The ability of the test substance to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analysed was selected based on the solubility of the test substance in the culture medium.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range (see Appendix Ill). The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range (see Appendix IV). The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9-mix the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments..

No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Executive summary:

The ability of the test substance to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analysed was selected based on the solubility of the test substance in the culture medium.

The number of cells with chromosome  aberrations found in the solvent control cultures was within the laboratory historical control data range (see Appendix Ill). The number of polyploid cells and cells with endoreduplicated chromosomes  in the solvent control cultures was within the laboratory historical control data range (see Appendix IV). The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9-mix the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome  aberrations in two independent experiments..

No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome  aberrations under the experimental conditions described in this report.