Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES Test: Negative; OECD 471; Zeiger, et al., (1987)

in vitro mammalian chromosome aberration test: Positive; OECD 473; Pritchard (2013)

in vitro mammalian cell gene mutation test: Negative; OECD 476; Woods (2013)

Link to relevant study records

Referenceopen allclose all

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.
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
Target gene:
Not applicable
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)
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.
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'.
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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 February 2013 to 02 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Recent Guideline study conducted to GLP
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK+/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y mouse lymphoma (3.7.2c) cells
Additional strain / cell type characteristics:
not applicable
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:
1.11, 2.23, 4.46, 8.92, 17.84, 35.68, 71.36, 142.73, 285.45 and 570.9 ug/mL (preliminary test
0.25, 1, 2.5, 10, 20 ug/mL (3 hours without S9 mix)
0.25, 2.5, 25, 50 ug/mL (3 hours with S9 mix)
0.25, 0.5, 1, 1.75, 2.5 ug/mL (24 hours without S9 mix)
Vehicle / solvent:
water (purified by reverse osmosis)
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
Cultures contained a total of 1.2 x 107 cells in a final volume of 10 mL. The final concentration of the S9 fraction was 2% v/v, if present. Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls. Aliquots of 100 uL of test substance dilution (at 100 times the desired final concentration), vehicle or positive control were added, then all cultures were incubated, with continuous shaking, for 3-hours at 37C. At least four serial dilutions of the test substance were tested.
Following the 3-hour exposure, the cells were washed once, resuspended in R10p to nominally 2 x 105 cells/mL (assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24-hours the cell density was readjusted to 2 x 105 cells/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 105 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cultures that were assessed were chosen at the Study Director’s discretion. Cloning efficiency was assessed by plating 1.6 cells/well in R20p, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 103 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 37C in an atmosphere of 5% CO2 in air.

After the plates had been incubated for at least 7 days for viability plates and approximately 10 to 14 days for mutant plates, the number of empty wells was assessed for each 96 well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF). The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants. The maximum concentrations assessed for mutant frequency in the main test was 20 and 50 ug/mL in the absence and presence of S9 mix respectively.

A test with a 24-hour exposure in the absence of S9 mix was carried out. Duplicate 10 mL cultures containing 3 x 106 cells were treated for 24-hours with 100 uL of test substance or positive control. Quadruplicate cultures were prepared for vehicle controls. At the end of the exposure period, the cells were washed once, resuspended in 10 mL R10p and counted to ascertain treatment growth. The cultures were then diluted to 2 x 105 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24-hours the cell density was readjusted to 2 x 105 cells/mL with R10p, the intention being to retain at least 1 x 107 cells. Following this, the procedure was the same as in the 3-hour treatment. The maximum concentration assessed for mutant frequency in the main test was 2.5 ug/mL.
Evaluation criteria:
Tests were accepted on the basis of the following criteria:

Acceptance criteria for test substance:

The highest concentration tested was one that allowed the maximum exposure up to 5000 ug/mL or 10 mM for freely soluble compounds, or the limit of toxicity (ie. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.

Acceptance criteria for vehicle controls:
The mean vehicle control value for mutant frequency was between 50 to 170 x 10-6.
The mean cloning efficiency was between 65 to 120%.
The mean suspension growth was between 8 to 32 on Day 2 following 3-hour treatments and between 32 to 180 on Day 2 following a 24-hour treatment.

Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining.

Acceptance criteria for positive controls:
Positive controls showed an absolute increase in mean total MF above the mean concurrent vehicle control MF of at least 300 x 10-6. At least 40% of this was due to the number of small mutant colonies.
Mean RTG’s for the positive controls were greater than 10%.

There was an absence of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity.
There was not excessive heterogeneity between replicate cultures.
Assays that did not fulfil the required criteria were rejected and therefore are not reported. This decision was at the discretion of the Study Director.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989) using a one-sided F-test, where p<0.001. Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: L5178Y mouse lymphoma (3.7.2c) cells
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that Allylamine did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.
Executive summary:

Allylamine was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3-hours or 24-hours in the absence of exogenous metabolic activation (S9 mix) or 3-hours in the presence of S9 mix.

Allylamine was found to be soluble at 57.09 mg/mL in water. A final concentration of

570.9 g/mL, dosed at 1%v/v (10 mM), was used as the maximum concentration in the preliminary toxicity test, in order to test up to the standard limit concentration within this test system as recommended in the regulatory guidelines.

Toxicity was observed in the preliminary toxicity test. Following a 3-hour exposure to allylamine at concentrations from 1.11 to 570.9 g/mL, relative suspension growth (RSG) was reduced from 100 to 10% and from 79 to 17% in the absence and presence of S9 mix respectively. Following a 24-hour exposure in the absence of S9 mix RSG was reduced from 96 to 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG).

Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix were 20 and 50 g/mL respectively. In the absence and presence of S9 mix RTG was reduced to 15 and 21% respectively.

In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 2.5 g/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The RTG was reduced to 25%.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

It was concluded that allylamine did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Although not conducted to GLP, this study was conducted equivalent to OECD Guideline 471. One deficiency in the study was that only four strains of bacteria were tested, and a fifth designed to detect cross-linking mutagens (e.g. TA102, E. coli WP2) was not included. Regardless of this deficiency, the available data in this study is suitable for assessing the mutagenicity of the substance in bacteria as it is not mandatory to test a species for cross-linking mutagens.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only 4 strains tested
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Liver S-9 Fraction (Hamster/Rat)
Test concentrations with justification for top dose:
5 doses of the substance were tested in triplicate. The actual test concentrations for this study were not presented in the source literature. The concentrations tested were based on the results of a range-finding toxicity study.
Vehicle / solvent:
Distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine; 2-aminoanthracene
Evaluation criteria:
A substance was judged mutagenic if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic if a low-level dose response was seen. A chemical was judged to be mutagenic, or weakly mutagenic, if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Raw Data

a) TA100

 Dose     Raw     10 % HLI     10 % RLI
 μg/Plate  Mean  Standard Error  Mean  Standard Error  Mean  Standard Error
 0  152  0.6  165  15.9  145  14.4
 1  -  -  -  -  -
 3  -  -  -  -  -  -
 10  -  - 133   3.0  -  -
 33  148  6.2  152  15.2  151  15.1
 100 141   5.0  115  9.2  149  5.0
 333  154  11.5  111  9.4  120  6.0
 1000  141  4.1  0  0.3  128  7.6
 3333  2  1.7  -  -  14  3.7
 POS  1730  33.0  2633  99.4  2457  78.1

HLI - Hamster Liver S9

RLI - Rat Liver S9

POS - Positive Control

b) TA1535

Dose     Raw     10 % HLI     10 % RLI
 μg/Plate  Mean  Standard Error  Mean  Standard Error  Mean  Standard Error
 0 20 1.5 10 1.3 11  3.5
 1 23 1.9
 3 24 1.8
 10 23 2.0 10 2.0 8 0.9
 33 26 1.7 7 1.3 9 1.2
 100 19 1.7 6 1.7 12 3.5
 333 8 0.9 6 1.9
 1000   6 1.5 10 0.6
 3333            
 POS  1130  33.6  340  11.7  257  1.9

HLI - Hamster Liver S9

RLI - Rat Liver S9

POS - Positive Control

c) TA1537

Dose     Raw     10 % HLI     10 % RLI
 μg/Plate  Mean  Standard Error  Mean  Standard Error  Mean  Standard Error
 0 5 1.2 10 1.5 10  1.9
 1 - - - - - -
 3 - - - - - -
 10 3 0.9 9 2.8 7 0.3
 33 3 1.0 9 1.7 6 1.5
 100 6 1.2 13 2.1 9 1.7
 333 3 0.9 8 1.5 8 1.0
 1000  4 1.8 6 1.7 11 1.5
 3333  -  -  -  -  -  -
 POS  755  300.7  139  17.0  157  18.4

HLI - Hamster Liver S9

RLI - Rat Liver S9

POS - Positive Control

d) TA98

    Raw     10 % HLI     10 % RLI
 μg/Plate  Mean  Standard Error  Mean  Standard Error  Mean  Standard Error
 0 21 3.5 18 1.5 18 2.7
 1
 3
 10 20 3.2
 33 19 1.3 19 2.9 16 1.3
 100 18 1.2 21 4.4 19 1.2
 333 14 1.2 21 1.2 13 1.2
 1000  16 3.2 17 2.2 20 2.1
 3333 7 2.2 16 1.9
 POS 271 17.9 1318 49.2 1421 127.8 

HLI - Hamster Liver S9

RLI - Rat Liver S9

POS - Positive Control

Conclusions:
Interpretation of results (migrated information):
negative

Allylamine produced negative results for bacterial reverse mutation both in the presence and absence of metabolic activation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

During an in vivo micronucleus study (Barfield, 2013) on allylamine, no evidence of an increase in the frequency of micronucleated polychromatic erythrocytes was observed.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 January 2014 to 7 April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant study, performed following an appropriate international test guideline (OECD 474).
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
in rats
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
The test organisms were supplied by Charles River UK Limited, Margate, Kent, England. For the preliminary toxicity tests both male (26.3 to 31.2 g) and female (21.2 to 25.3 g) animals were tested. During the definitive stage of the study male mice 35 to 45 days old and weighting 22.2 g and 27.6 g were used. Upon arrival at the test facility the weight of the animals was checked and found to be acceptable. The animals were randomly assigned to groups and given a unique tail tattoo. Each group was kept with the sexes separated in cages. The animals were kept in a controlled environment with the thermostat and relative humidity within target ranges of 19 to 23°C and 40 to 70% respectively, throughout the study. The room was illuminated by artificial light for 12 hours per day.

All animals were allowed free access to pelleted expanded rat and mouse No.1 maintenance diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK) and tap water ad libitum. Animals were acclimatised to laboratory conditions for at least 5 days before testing.
Route of administration:
oral: gavage
Vehicle:
Suspensions of the test substance were prepared in 1% methylcellulose in purified water. Methylcelluose was obtained from Sigma Aldrich batch number SLBG2263V.
Details on exposure:
From the results obtained in the preliminary toxicity test, dose levels of 18.75, 37.5 and 75 mg/kg/day were used for the micronucleus test. Refer to Table 1 for details of the study design. All animals in the vehicle control, test substance dose groups were dosed orally by gavage, on two occasions approximately 24 hours apart, using a dose volume of 10 mL/kg. The positive control group were dosed at 20 mL/kg via oral gavage.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
Animals were treated with allylamine orally by gavage on two occasions approximately 24 hours apart.
Post exposure period:
24 hours
Remarks:
Doses / Concentrations:
18.75, 37.5 and 75 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
The definitive study was performed on males only, as no significant differences in toxicity were observed between the sexs, during the preliminary toxicity study. Please refer toTable 1 for numbers of animals per treatment.
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C obtained from Sigma Aldrich, batch number SLBD1982V was used as the positive control compound. A solution was prepared using purified water at a concentration of 0.6 mg/mL just prior to administration. Animals in the positive control group were dosed on a single occasion at 20 mL/kg via oral gavage.
Tissues and cell types examined:
Slides prepared from the bone marrow of both femurs of each animal were evaluated for presence of micronucleated polychromatic and normochromatic erythrocytes.
Details of tissue and slide preparation:
The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration. The resulting cell suspensions were centrifuged at 1000 rpm (150 x g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum and the following fixation and staining procedure was followed; The suspensions were firstly fixed for a minimum of 10 minutes in methanol and allowed to air-dry. After which they were rinsed in purified water and stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes. After staining the suspensions were washed in purified water for 5 minutes, rinsed in cold tap water for 2 minutes and stored at room temperature until required. Immediately prior to scoring, slides are wet mounted with coverslips using purified water.
Evaluation criteria:
Coded slides were examined by fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.
Statistics:
For the proportion of polychromatic erythrocytes at 24 hours after dosing, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.

For incidences of micronucleated polychromatic erythrocytes at 24 hours after dosing, an exact one-tailed Linear-by-Linear association test (Cytel 1995) with “step-down” was used on Groups 1 to 4 for an increase from control. If significant, then the analysis was carried out on Groups 1 to 3. Exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No clinical signs of toxicity were observed for the vehicle control, positive control or animals administered Allylamine at any treatment level over the duration of the test. Some small incidences of bodyweight loss were observed throughout the micronucleus test.

Allylamine did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes in male CD1 mice. Allylamine did not cause any significant increases in the incidence of micronucleated normochromatic erythrocytes in male CD1 mice. Allylamine did not cause any statistically significant decreases in the proportion of polychromatic erythrocytes in male CD1 mice.

Table 2: Individual animal data

 

Treatment              (mg/kg/day)

Animal number

Proportion PCE (%)

MPCE

PCE

NCE

MNCE

Vehicle

201

54.3

3

546

459

0

 

202

37.1

1

380

644

0

 

203

39.3

1

393

607

0

 

204

49.0

1

491

512

0

 

205

43.0

2

432

573

0

 

206

61.0

0

662

423

0

Allylamine

211

45.0

1

452

553

0

(18.75)

212

49.0

1

490

511

0

 

213

49.0

2

490

511

0

 

214

58.9

2

594

415

0

 

215

62.9

0

633

374

0

 

216

47.7

1

480

527

0

Allylamine

221

54.7

0

551

456

0

(37.5)

222

43.2

1

440

578

0

 

223

49.1

1

518

538

0

 

224

51.3

1

520

494

0

 

225

44.2

2

445

562

0

 

226

43.8

2

440

564

0

Allylamine

231

48.6

2

487

515

0

(75)

232

41.4

2

415

587

0

 

233

47.1

2

473

531

0

 

234

45.7

2

461

548

0

 

235

48.0

1

481

522

0

 

236

50.3

1

505

499

0

 

237

47.4

1

479

531

0

 

238

49.9

2

508

510

0

MitomycinCa

 

241

54.0

32

554

471

0

(12)

242

37.0

47

372

633

0

 

243

45.2

43

457

554

0

 

244

49.4

37

495

508

0

 

245

56.3

39

578

448

0

 

Vehicle

1% methylcellulose in purified water.

PCE

Polychromatic erythrocytes

MPCE

Number of micronucleated cells observed per 2000 polychromatic erythrocytes examined

NCE

Total number of normochromatic erythrocytes examined for micronuclei

MNCE

Number of micronucleated normochromatic erythrocytes observed

a

Positive control dosed once only 24 hours prior to termination

Conclusions:
Interpretation of results (migrated information): negative
Under the conidtions of this study, orally dosed allylamine did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male CD1 mice.
Executive summary:

A study was performed to assess the potential induction of micronuclei by allylamine in bone marrow cells of CD1 mice. Animals were treated with allylamine orally by gavage on two occasions approximately 24 hours apart. On the basis of results a preliminary toxicity test, dose levels of 18.75, 37.5 and 75 mg/kg/day were selected for the micronucleus test. No substantial differences in toxicity were observed between the sexes in the preliminary toxicity test, therefore, the micronucleus test was performed using male animals only. Analysis of bone smears collected 24 hours after the second treatment revealed no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes and no statistically significant decreases in the proportion of polychromatic erythrocytes at any dose level, compared to vehicle control values.The positive control compound, Mitomycin C, produced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (p<0.01).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

During an in vitro test (Pritchard, 2013), the test material was shown to cause an increase in the frequency of structural chromosome aberrations in the absence of S9 mix following 3-hour treatment, at 350 µg/mL only. An in vivo chromosome aberrations study was therefore performed (Barfield, 2013). This study concluded that the test substance did not cause an increase in the induction of micronucleated polychromatic erythrocytes in CD1 mice when administered orally by gavage up to a dose of 75 mg/kg/day.


Justification for selection of genetic toxicity endpoint
In vivo study, triggered by positive result from one in vitro study.

Justification for classification or non-classification

In accordance with Regulation (EC) No 1272/2008 (CLP) allylamine was non-classified in regards to genetic toxicity, based on a negative result from an in vivo study.