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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 February 2013 to 23 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was conducted in accordance with international guidelines (OECD 473), and in compliance with the UK GLP Regulations. All relevant criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identity: Allylamine

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
mammalian cell line, other: human lymphocytes
Details on mammalian cell type (if applicable):
In this study, human blood was taken from two healthy, non-smoking, adult donors, pooled and diluted with tissue culture medium. The cultures were incubated in the presence of PHA before being treated with the test substance.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix, dosed with phenobarbital and 5,6 benzoflavone
Test concentrations with justification for top dose:
0, 100, 300 and 350 ug/mL (3 hour, without S9 mix)
0, 100, 200 and 570.9 ug/mL (3 hour, with S9 mix)
Vehicle / solvent:
water (purified in-house by reverse osmosis)
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
For 3 hours without S9 mix, Allylamine was added to each culture in 50 µL aliquots to give final concentrations of 100, 200, 250, 300, 350, 400 and 570.9 µg/mL. Water was used as the vehicle control, and Mitomycin C at a final concentration of 0.2 µg/mL was the positive control.

For 3 hours with S9 mix, Allylamine was added to each culture in 50 µL aliquots to give final concentrations of 100, 200, 300, 400 and 570.9 µg/mL. Water was used as the vehicle control, and Cyclophosphamide at a final concentration of 5 µg/mL was the positive control.

For 21 hours without S9 mix, Allylamine was added to each culture in 50 µL aliquots to give final concentrations of 50, 75, 100, 150, 200, 225, 250, 275, 300 and 325 µg/mL. Water was used as the vehicle control, and Mitomycin C at a final concentration of 0.1 µg/mL was the positive control.

Only the following were evaluated:
0, 100, 300 and 350 ug/mL (3 hour, without S9 mix)
0, 100, 200 and 570.9 ug/mL (3 hour, with S9 mix)

Evaluation criteria:
An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.
Statistics:
The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973).
A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.

D20’s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated (where possible) using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002). The following model was used:
p = C + (1 - C)/1 + exp{– intercept – slope ln(conc)}

p is the proportion of cells with aberrations, conc is the concentration of test substance. C is a parameter estimating the control proportion of aberrations.

The D20 values for structural aberrations (including and excluding gaps) are retained within the study data.

The data was analysed using the SAFEStat (SAS statistical applications for end users, version 1.1) Chromosome Aberrations application (version 1.0) which was developed in SAS (SAS INSTITUTE 2002).

As a clear statistically significant response was observed in the absence of S9 mix, with mean values which exceeded the laboratory historical control range, no further testing was conducted.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Only in absence of S9 mix
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: other: cultured human lymphocytes
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

It is concluded that Allylamine has shown evidence of causing an increase in the frequency of structural chromosome aberrations in the absence of S9 mix following 3-hour treatment, at 350 µg/mL only, in this in vitro cytogenetic test system, under the experimental conditions described.
Executive summary:

A study was performed to assess the ability of allylamine to induce structural chromosome aberrations in human lymphocytes cultured in vitro.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Vehicle and positive control cultures were also included. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

A preliminary toxicity test was performed in order to determine the toxicity of allylamine to cultured human lymphocytes. A 3-hour treatment in the absence and presence of S9 mix, and a 21-hour continuous treatment in the absence of S9 mix were used to determine toxicity.

In the main test, the mitotic index was assessed for all cultures treated with allylamine and the vehicle control, water (purified in-house by reverse osmosis). Justification for the highest analysed concentration was based on the limit final concentration (10 mM) for this test system where no significant reduction in mitotic index greater than 50% was observed. Otherwise, selection was determined by cytotoxicity.

On the basis of these data, the following concentrations were selected for metaphase analysis: In the absence of S9 mix, 3-hour treatment: 100, 300 and 350 μg/mL.

In the presence of S9 mix, 3-hour treatment: 100, 200, 400 and 570.9 μg/mL.

In the absence of S9 mix following 3-hour treatment, allylamine caused statistically significant increases (p<0.001: including and excluding gaps) in the proportion of metaphase figures containing chromosomal aberrations at 350 μg/mL only, when compared to the vehicle control. These increases were reproducible between duplicate cultures, and within an acceptable level of toxicity, with mean values that exceeded the laboratory historical control range, when taken at the 99% confidence limit. At the lower analysed treatment concentrations of 100 and 300 μg/mL, no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations were observed, when compared to the vehicle control, with mean values within laboratory historical control range, when taken at the 99% confidence limit. All mean values for the vehicle control (water) were within laboratory historical control range, when taken at the 99% confidence limit.

In the presence of S9 mix following 3-hour treatment, allylamine caused statistically significant increases (p<0.001: including and excluding gaps) in the proportion of metaphase figures containing chromosomal aberrations at 570.9 μg/mL only, the limit guideline concentration, when compared to the vehicle control. These increases were within or acceptably close to the laboratory historical control range when taken at the 99% confidence limit. At the lower analysed treatment concentrations of 100 and 200 μg/mL, no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations were observed, when compared to the vehicle control, with mean values within laboratory historical control range, when taken at the 99% confidence limit. All mean values for the vehicle control (water) were within laboratory historical control range, when taken at the 99% confidence limit.

However, due to the disparity between replicate mitotic index values at 570.9 μg/mL, an additional lower treatment concentration of 400 μg/mL was analysed to establish if the observed response at 570.9 μg/mL was biologically relevant. Furthermore, toxicity at

400 μg/mL was closer to guideline limit for toxicity. At 400 μg/mL, mean values were within laboratory historical control range, when taken at the 99% confidence limit. Therefore, the apparent increases at 570 μg/mL were considered biologically non-relevant, as values (including and excluding gaps) were within or acceptably close to the laboratory historical control range, when taken at the 99% confidence limit, yet within the historical control range, with no associated concentration-related response. In addition, the statistical significance had been exaggerated by the low vehicle control values.

No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, under any treatment condition, when compared to the vehicle control.

As a clear statistically significant response was displayed in the absence of S9 mix following 3-hour treatment at the 350 μg/mL only, with mean values which clearly exceeded the laboratory historical control range, no further metaphase analysis (of cultures treated for 21-hour continuously in the absence of S9 mix) was conducted.

Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

It is concluded that allylamine has shown evidence of causing an increase in the frequency of structural chromosome aberrations in the absence of S9 mix following 3-hour treatment, at 350 μg/mL only, in this in vitro cytogenetic test system, under the experimental conditions described.