Diallylamine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 24 July 2013 and 19 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Mammalian somatic cell cytogenetic assay

Test material

Test material information

Constituent 1

Reference substance name:

Diallylamine

EC Number:

204-671-2

EC Name:

Diallylamine

Cas Number:

124-02-7

Molecular formula:

C6H11N

IUPAC Name:

diallylamine

Test material form:

liquid

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Hsd:ICR (CD-1) mice were received from Harlan on 30 Jul 2013 in the dose range-finding assay (DRF) and 22 Aug 2013 in definitive assay. A second set of animals was received for bioanalysis on 07 May 2014.
At the time of dose adminstration, the mice were six weeks old in all assays.
Body weights of mice assigned to the study groups at randomization were within the following ranges:
Dose range-finding assay: Males 31.6 - 35.9 g; Females 25.4 - 30.4 g
Definitive assay: Males 32.0 - 37.0 g; Females 23.4 - 29.1 g
Bioanalysis: Males 33.3 - 36.7 g; Females 26.1 - 31.1 g

The animals were acclimated for 6 days in the DRF, 5 days in the definitive study and 6 days in bioanalysis.

ENVIRONMENTAL CONDITIONS
Animals were housed in a controlled environment at 72 ± 3°F and 50 ± 20% relative humidity with a 12-hour light/dark cycle. The light cycle may have been interrupted for study related activities. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air per hour.

Animals of the same sex were housed up to five per Micro-Barrier cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system.

BEDDING, FOOD AND WATER
Heat treated hardwood chips were used for bedding to absorb liquids.
A certified rodent chow was provided ad libitum.
Animals had free access to tap water.
There were no contaminants in the bedding, feed and water that were expected to interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Deionized water was selected as the solvent

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The vehicle used was Deionized water. Dose formulations were prepared prior to dose administration as follows:
An appropriate amount of test article was added into an amber vial. An appropriate amount of vehicle was added and the formulation was mixed magnetically until homogenous in appearance. All formulation appeared as colorless liquids or solutions.

Dose Administration and Observation:
All dose formulations were administered at a volume of 10 mL/kg by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles).
Body weights were recorded prior to the first dose for the purpose of dose volume calculations in both assays. Animals were also weighed on the day of sacrifice in the DRF assay. Animals were observed pre-dose, one and two hours following dose adminstration and at least once daily thereafter for clinical signs of toxicity in both assays, excluding animals reserved for bioanalysis.

Duration of treatment / exposure:

See dose adminstration and observation.

Frequency of treatment:

Single oral dose administration.

Post exposure period:

24 and 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Definitive assay - male
50 mg/kg (Group 2)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Definitive assay - male
100 mg/kg (Group 3)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Definitive assay - male
200 mg/kg (Group 4)

Doses / concentrations 4

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

Definitive assay - females
75 mg/kg (Group 2)

Doses / concentrations 5

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Definitive assay - females
150 mg/kg (Group 3)

Doses / concentrations 6

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Definitive assay - females
300 mg/kg (Group 4)

Doses / concentrations 7

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Definitive assay - vehicle (Group 1)

Doses / concentrations 8

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Definitive assay - positive control (Cyclophosphamide) (Group 6)

No. of animals per sex per dose:

The definitive assay dose levels tested were 50, 100, and 200 mg/kg in male mice and 75, 150 and 300 mg/kg in female mice. Groups 1 and 4 consisted of 10 animals/sex/group designated for either 24 or 48 hour bone marrow collection and Groups 2, 3 and 5 consisted of 5 animals/sex/group designated for 24 hour bone marrow collection. Five additional animals/sex were added to Group 4 in the event of unexpected mortality.

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide;
The positive control was an aqueous dosing formulation of cyclophosphamide, at a concentration of 5 mg/mL, that was prepared fresh on the day of dose administration. An appropriate amount of CP was dissolved in an appropriate volume of sterile water for injection. The formulation appeared as a clear colorless solution.

Examinations

Tissues and cell types examined:

polychromatic erythrocyte (PCE) cells in mouse bone marrow.

Details of tissue and slide preparation:

BLOOD COLLECTION AND SAMPLE HANDLING
Design:
Satellite groups of three animals/sex/group were dosed with the vehicle (Group 6) or test item at 50 (Group 7), 100 (Group 8) , and 200 (Group 9) mg/kg in male mice or 75 (Group 7), 150 (Group 8) and 300 (Group 9) mg/kg in female mice. Animals administered with the vehicle control were bled one hour post-dose and animals dosed with the test article were bled one and four hours post-dose.

Another satellite group of animals were dosed with the vehicle (Group 10) or test item at 50 (Group 11) mg/kg (males) or 75 (Group 11) mg/kg (females). Animals were bled one hour post-dose.

See satellite group dosing and regimen tables in any other information on material incl. tables section.

SAMPLE COLLECTION AND STORAGE
The animals were anesthetized prior to blood collection by exposure to 70% CO2/30% O2. Each sample (0.5 mL) was collected via retro-orbital sinus into a single, pre-labeled K3EDTA tube and inverted several times to ensure adequate mixing of blood and anticoagulant. Blood samples were placed on wet ice pending centrifugation. Within 60 minutes of collection, samples were centrifuged for 5 minutes at 1000 g, 2-8 ºC. Plasma was harvested into two approximately equal aliquots.

Following blood collection, animals were euthanized by CO2 asphyxiation and discarded without further examination.

Plasma samples from the following groups were stored at ≤ -60 ºC until packed on dry ice and transferred to the BioReliance Analytical Chemistry Department for analysis:

• Group 6 (males, 1 hour collection timepoint; primary)

• Group 9 (males & females; 1 hour collection timepoint; primary)

• Group 10 – 11 (males & females; one hour collection timepoint; primary)

BONE MARROW COLLECTION AND SLIDE PREPARATION
Femoral bone marrow was collected at approximately 24 or 48 hours after the final dose in the definitive assay and after the two day observation period in the DRF. Animals were euthanized by carbon dioxide inhalation. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow was transferred to a centrifuge tube containing 1 mL fetal bovine serum. The cells were pelleted by centrifugation, and the supernatant was drawn off leaving a small amount of fetal bovine serum with the pellet. Cells were re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. At least two slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of slides was stained with acridine orange for microscopic evaluation, the other was reserved as a backup. Each slide was identified by the harvest date, study number, and animal number. Slides were coded using a random number table by an individual not involved with the scoring process.

SCORING AND STATISTICAL ANALYSIS
Bone marrow was evaluated by fluorescent microscopy. The staining procedure permits the differentiation by color of polychromatic and normochromatic erythrocytes (bright orange PCEs and ghost-like, dark green NCEs, respectively). The criteria for the identification of micronuclei are those of Schmid (1975). Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. Scoring was based upon the micronucleated cell, not the micronucleus; thus, occasional cells with more than one micronucleus were counted as one micronucleated PCE (mnPCE), not two (or more) micronuclei. At least 2000 PCEs/animal were scored for the presence of micronuclei (mnPCEs) whenever possible. In addition, at least 1000 total erythrocytes (PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity. PCE/EC proportions <20% of vehicle control value were considered excessively cytotoxic and the animal data was excluded from evaluation. The frequency of mnPCEs and the proportion of PCEs to total erythrocytes was determined for each animal and treatment group. Statistical significance (p ≤ 0.05) was determined using the binomial distribution (Kastenbaum-Bowman tables).

Evaluation criteria:

Criteria for a valid test:
The vehicle control group should have been consistent with the historical vehicle control range, and must have been ≤ 0.4% mnPCEs (Akihiro et al., 1998) and the positive control must have induced a significant increase (p ≤ 0.05) in mnPCE frequency as compared to the concurrent vehicle control.
Five animals/sex/group were available for analysis.

Evaluation of Test Results:
Once the criteria for a valid assay were met, the results were evaluated. Test article was considered to be positive if it induced a significant increase in mnPCE frequency (p ≤ 0.05) at any dose level or sampling time compared to the concurrent vehicle control. The test article was considered to be negative if no significant increase in mnPCE frequency was observed (p > 0.05) compared to the concurrent vehicle control. Other criteria may have been used in reaching a conclusion about the study results (e.g. magnitude of any increase, dose-dependency, comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment to clearly report and describe any such considerations.

Statistics:

Statistical significance (p ≤ 0.05) was determined using the binomial distribution (Kastenbaum-Bowman tables).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

No statistically significant increase in the number of micronucleated polychromatic erythrocytes observed in the test article groups compared to the vehicle control group at 24 or 48 hours post-dosing.

Toxicity:

yes

Remarks:

Minor clinical signs (toxicity) observed. The test did not induce cytotoxicity.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose Range-Finding Assay:
Animals dosed with the vehicle and with the test article at 100 mg/kg appeared normal throughout the study. Animals dosed with the test item at 200 mg/kg displayed piloerection while animals dosed at 300 mg/kg exhibited lethargy, piloerection, hunched position, crusty eyes and mortality (3/3 males). Animals dosed at 400 mg/kg displayed lethargy, prostration, piloerection, hunched position, crusty eyes, crusty nose, irregular breathing and mortality (2/3 males and 3/3 females). At the 500 mg/kg dose level, males displayed lethargy, piloerection, prostration, hunched position, crusty eyes, crusty nose and irregular breathing while all female animals were found dead one hour post-dose administration.

No reductions in the ratio of PCEs to total erythrocytes in the test article groups compared to the respective vehicle control groups were observed. No statistically significant increase in the incidence of mnPCEs in the test article-treated groups relative to the respective vehicle control group was observed either (p < 0.05, Kastenbaum-Bowman tables).

Definitive Micronucleus Assay:
No mortality occurred at any dose level during the course of the definitive assay. Clinical signs included lethargy and piloerection in male mice at 200 mg/kg and lethargy, piloerection, hunched position and crusty eyes in female mice at 300 mg/kg. All other mice appeared normal throughout the study.

Bone Marrow Analysis in the Definitive Micronucleus Assay:
No appreciable reductions in the PCEs/EC ratio in the test article groups compared to the vehicle control group were observed indicating the test did not induce cytotoxicity.

No statistically significant increase in the incidence of mnPCEs in the test-article treated groups was observed relative to the negative control group (p > 0.05, Kastenbaum-Bowman tables).
The positive control, CP, induced a statistically significant increase in the incidence of mnPCEs (p < 0.05, Kastenbaum-Bowman tables).
The number of mnPCEs in the vehicle control groups did not exceed the historical control range.

The incidence of mnPCEs per 10,000 PCEs scored (2000 PCEs/mouse) and the proportion of polychromatic erythrocytes per total erythrocytes are summarized (see table in any other information on results incl. tables)

Based upon this, all criteria for a valid test were met.


BioAnalysis:
Plasma samples were analyzed for concentrations of the test item. Measurable concentrations of the test item were detected in the plasma samples of the test article dosed groups. Plasma samples from the vehicle control group showed no quantifiable levels of the test item.

Any other information on results incl. tables

Table:Summary of BoneMarrow Micronucleus Analysis Followinga Single Oral Administration of the test article in Male and Female Hsd:ICR(CD-1) Mice in the Definitive Assay

 

		Time	Number of	PCE/Total Erythrocytes			Change from	Numberof MPCE/1000 PCE			Number of
Treatment(10mL/kg)	Sex	(hr)	Animals	(Mean +/-SD)			Control (%)	(Mean +/-SD)			MPCE/PCE Scored
DeionizedWater
	M	24	5	0.481	±	0.04	---	0.5	±	0.50	5	/	10000
	F	24	5	0.512	±	0.03	---	1.0	±	0.35	10	/	10000
Test article
50 mg/kg	M	24	5	0.472	±	0.04	-2	0.8	±	0.27	8	/	10000
75 mg/kg	F	24	5	0.506	±	0.03	-1	0.8	±	0.27	8	/	10000
100 mg/kg	M	24	5	0.494	±	0.02	3	0.4	±	0.22	4	/	10000
150 mg/kg	F	24	5	0.509	±	0.04	-1	1.0	±	0.00	10	/	10000
200 mg/kg	M	24	5	0.451	±	0.06	-6	0.5	±	0.00	5	/	10000
300 mg/kg	F	24	5	0.490	±	0.02	-4	0.8	±	0.27	8	/	10000
Cyclophosphamide
50 mg/kg	M	24	5	0.472	±	0.01	-2	21.7	±	3.09	*217	/	10000
	F	24	5	0.490	±	0.03	-4	16.7	±	3.27	*167	/	10000
DeionizedWater
	M	48	5	0.470	±	0.06	---	0.6	±	0.22	6	/	10000
	F	48	5	0.497	±	0.06	---	0.8	±	0.27	8	/	10000
Test article
200 mg/kg	M	48	5	0.485	±	0.04	3	0.4	±	0.42	4	/	10000
300 mg/kg	F	48	5	0.523	±	0.02	5	0.6	±	0.22	6	/	10000

*p≤0.05(Kastenbaum-BowmanTables)

PCE–polychromaticerythrocytes

 

Mouse Micronucleus Test Historical Control Data

Negative Control

 

Parameter	Ratioof PCE/TotalErythrocytes		Numberof mnPCE/2000PCEScored/Animal		Numberof mnPCE/10000 PCEScored/Group
	Males	Females	Males	Females	Males	Females
Mean	0.53	0.55	0.58	0.65	2.90	3.25
StandardDeviation	0.06	0.07	0.71	0.76	2.30	2.24
Range	0.37-0.65	0.33-0.70	0-3	0-2	0-7	0 8

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met. The results of the micronucleus assay indicate that the test item did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes. Measurable concentrations of test item were detected in the plasma samples of the test article dosed groups. Therefore, the test item was concluded to be negative.

Executive summary:

The test article was evaluated for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in mouse bone marrow. Deionized water was selected as the solvent. Test and/or control article formulations were administered at a dose volume of 10 mL/kg by oral gavage.

In the dose range finding assay (DRF), the maximum dose tested was 500 mg/kg. The other dose levels tested were 100, 200, 300 and 400 mg/kg in three animals/sex/group. Based upon the results, the high dose selected in the definitive assay was 300 mg/kg in female mice and 200 mg/kg in male mice.

The definitive assay dose levels tested were 50, 100, and 200 mg/kg in male mice and 75, 150 and 300 mg/kg in female mice. Groups 1 and 4 consisted of 10 animals/sex/group designated for either 24 or 48 hour bone marrow collection and Groups 2, 3 and 5 consisted of 5 animals/sex/group designated for 24 hour bone marrow collection. Five additional animals/sex were added to Group 4 in the event of unexpected mortality. Following scheduled euthanasia, femoral bone marrow was collected; bone marrow slides were prepared and stained with acridine orange. Bone marrow cells [polychromatic erythrocytes (2000 PCEs/animal)] were examined microscopically for the presence of micronuclei (micronucleated PCEs; mnPCEs) and statistical analysis of data was performed using the Kastenbaum-Bowman Tables (binomial distribution, p ≤ 0.05). The ratio of polychromatic erythrocytes (PCEs) to total erythrocytes (EC) in the test article groups relative to the vehicle control groups was also evaluated to reflect the test article’s cytotoxicity.

Satellite groups of animals (three animals/sex/group) were dosed with the vehicle or test article at 50, 100, and 200 mg/kg in male mice or 75, 150 and 300 mg/kg in female mice. A second set of animals were dosed with the vehicle (3 males) or low dose (50 mg/kg in males and 75 mg/kg in females). Plasma samples from the vehicle (males) and low dose (males and females) from the first set of animals and plasma samples from the vehicle (males) and high dose (males and females) were retained at ≤ - 60 ºC and then transferred for analysis.

Measurable concentrations of the test item were detected in the plasma samples of the test article dosed groups. Plasma samples from the vehicle control group showed no quantifiable levels of test item. The results of the micronucleus assay indicate that the test item did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes. Under the conditions of this study, the administration of the test article at doses up to and including 200 mg/kg in male mice and 300 mg/kg in female mice was concluded to be negative in the micronucleus assay.