Diallylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity (in vitro mammalian chromosome aberration test) = Positive in the S9 activated treatment, OECD 473, BioReliance,

Genetic Toxicity (in vitro bacterial reverse mutation test) = Negative, equivalent to OECD 471, Zeiger et al

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 98%

Target gene:

histidine gene (for S. typhimurium)

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 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 3333 and 10,000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ET95, ETOH; 95% ethanol (solvent)

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

ET95

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min.
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: Tripicate (for each dose)

DETERMINATION OF CYTOTOXICITY
- Method: Number of his+ colonies and clearing in the density of background lawn.

Evaluation criteria:

An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic (+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose-related increase was judged insufficiently high to justify a call of "+W" if only a single dose was elevated over the control, or if a non-dose related increase was seen.

A chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials. A chemical was considered to be questionable (?) if a reproducible increase of his+ revertants did not meet the criteria for either a "+" or "+W", or if only single doses produced an increase in his+ revertants in repeat trials.

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

complete clearing of background lawn seen at 10000 µg/plate (TA100 and TA98) and at 3333 µg/plate (TA1537) without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

A summary of results obtained for diallylamine are presented below:

Mutagenic responses of Salmonella tester strains (mean ± SEM; three plate) to Diallylamine. The 0 µg/plate dose is the solvent control.

Abbreviations:

ET95, ETOH; 95% ethanol (solvent)

POS: Positive control

NA: Not acivated

RLI: Aroclor 1254 -induced rat liver S-9.

HLI: Aroclor 1254 -induced hamster liver S-9

t: complete clearing of background lawn (colonies not counted)

(-): non-mutagenic

Diallyamine

Solvent: ET95

Dose	TA100						TA1535						TA1537						TA98
µg/plate	NA (-)		10% HLI (-)		10% RLI       (-)		NA (-)		10% HLI (-)		10% RLI       (-)		NA (-)		10% HLI (-)		10% RLI       (-)		NA (-)		10% HLI (-)		10% RLI          (-)
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	86	5.1	146	5.3	115	12.5	5	1.2	10	1.3	7	0.6	7	0.7	9	0.9	6	0.7	14	4.5	26	1.8	25	1.5
33							5	2.7					7	2.3			5	0.6
100	88	4.7	139	3.4	136	9.8	5	1.8	16	0.6	8	0.6	6	0.3	10	1.2	5	1.2	17	1.9	19	1.5	18	3.2
333	94	2.5	138	9.5	130	9.2	5	0.7	10	0.9	5	0.9	5	1.5	8	0.7	7	0.6	18	0.9	22	1.7	21	1.5
1000	88	2.4	145	16.0	145	3.8	2	1.9	7	2.4	6	1.0	6	1.5	8	0.9	5	2.3	20	2.0	21	4.5	18	1.2
3333	87	3.2	135	5.8	143	12.4	2	1.2	7	1.8	6	1.2	t		5	0.9	2	0.6	7	3.1	15	4.1	17	2.6
10000	t		132	5.2	120	11.8	2	0.5	3	0.5	2	0.7			1	0.3			t		6	1.5	5	1.2
POS	441	41.9	1955	23.9	1186	54.6	532	19.8	359	64.3	344	29.9	199	57.9	162	5.2	268	30.4	197	9.3	1421	69.0	741	125.3

Conclusions:

Diallylamine was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Diallylamine was tested using a preincubation modification of the Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor induced male Sprague-Dawley rats and Syrian hamsters.

The tested Salmonella typhimurium strains were TA98, TA100, TA1535 and TA1537.

The test conceentrations of Diallylamine were 33, 100, 333, 1000, 3333 and 10,000 µg/plate, with concurrent solvent and positive controls.

Diallylamine was considered to be non-mutagenic under the conditions of this test. No reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials was observed.

The positive control chemicals used in thetest induced marked increases in the frequency of revertant colonies, both with and without metabolic activiation.

Complete clearing of background lawn was seen at 10000 µg/plate (TA100 and TA98) and at 3333 µg/plate (TA1537) without metabolic activation

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 15 April 2013 and 4 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: In vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

Test system:
Peripheral blood lymphocytes were obtained from a healthy non-smoking 26-year old adult female on 16 Apr 2013 for the preliminary toxicity assay and from the same donor on 30 Apr 2013 for the definitive assay. The donor has no recent history of radiotherapy, viral infection or the administration of drugs. This system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals.

Preparation of target cells:
Peripheral blood lymphocytes were cultered in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 µg/ml streptomycin) by adding 0.6 mL heparinized blood to a centrifuge tube containing 9.4 mL of complete medium with 1% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

Preliminary Toxicity Assay: 0.097 to 970 µg/mL
Chromosome Aberration Assay:
4 hr treatment (without activation): 75, 150, 300, 600, 970 µg/mL
4 hr treatment (with activation): 75, 150, 300, 600, 970 µg/mL
20 hr treament (without activation): 75, 150, 300, 600, 970 µg/mL

The dose levels selected for testing in the chromosome aberration assay were based on the results of the preliminary toxicity assay.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water used used as the vehicle based on the solubility information provided and compatibility with the target cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

CP was used dissolved and diluted in sterile distilled water to stock concentrations of 500 and 750 µg/mL (final concentrations of 5 and 7.5 µg/mL) for use as the positive control in the S9-activated test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

MMC was used dissolved and diluted in sterile distilled water to stock concentrations of 0.03 and 0.06 µg/mL (final concentrations of 0.3 and 0.6 µg/mL) for use as the positive control in the non-activated test system.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 44-48 hours
- Exposure duration: 4 hours (with and without activation), 20 hours (without activation)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 (duplicate)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
For the preliminary toxicity and the definitive assays, cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analyzed in the first division metaphase. Two hours prior to harvest, Colcemid was added to the cultures at a final concentration of 0.1 µg/mL.

Cells were collected by centrifugation, treated with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 3:1 v/v), capped and stored overnight or longer at 2-8°C. To prepare slides, the cells were collected by centrifugation and if necessary, the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with Giemsa, permanently mounted, and identified by the BioReliance study number, dose level, treatment condition, harvest date, activation system, test phase, and/or replicate tube design.

NUMBER OF CELLS EVALUATED:
100 per duplicate treatment (for structural and numerical aberrant cells)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
Whenever possible, a minimum of 200 metaphase spreads containing 46 centromeres from each dose level (100 per duplicate treatment) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The number of metaphase spreads that were examined and scored per duplicate culture may be reduced if the percentage of aberrant cells reaches a significant level (at least 10% determined based on historical positive control data) before 100 cells are scored.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;
The mitotic index was recorded as the percentage of cells in mitosis per 500 cells counted.

OTHER EXAMINATIONS:
- Determination of polyploidy:
The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated per 100 cells analyzed (when possible) for each culture (a total of 200 per dose level when possible).


- OTHER:

Evaluation criteria:

Evaluation of Test Results:
Toxicity induced by treatment is based upon inhibition of mitosis.
The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group.

A test article was considered positive if it induced a statistically significant and dose-dependent increase in the frequency of aberrant metaphases (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.

Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.).

Criteria for Determination of a Valid Test:
The frequency of cells with structural chromosome aberrations in the vehicle control group was within the historical range for vehicle controls. The percentage of cells with chromosome aberrations in the positive control group was statistically increased (p ≤ 0.05, Fisher's Exact test), relative to the vehicle control.

Statistics:

Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose- responsiveness.

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

structural aberrations

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

structural aberrations

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

numerical aberrations

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Solubility:
The test article was soluble in water at concentrations of 9.7 mg/mL, the maximum concentration tested in the study.

Preliminary Toxicity Assay:
A preliminary toxicity assay was conducted to observe the cytotoxicity profile of the test article and to select suitable dose levels for the definitive chromosome aberration assay. HPBL cells were first exposed to nine dose levels of R-50845, ranging from 0.097 µg/mL to 970 µg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor- induced S9 metabolic activation system for 4 hours, or continuously for 20 hours in the absence of S9 activation. The test article was soluble in water and in the treatment medium at all dose levels tested at the beginning and conclusion of the treatment period.

The osmolality in treatment medium of the highest dose level tested, 970 µg/mL, was 256 mmol/kg. The osmolality of the vehicle (water) in the treatment medium was 244 mmol/kg. The osmolality of the test article dose levels in the treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%. The pH of the highest dose level (970 µg/mL) of test article in treatment medium was measured to be 9.0. In order to maintain a neutral pH in the treatment medium, the pH was adjusted to 7.0 using 0.1 mL of 1N HCl.

Substantial toxicity (at least 50% reduction in mitotic index relative to the vehicle control) was not observed at any dose level in any of the three treatment groups. Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows:

4 hr treatment (without activation): 75, 150, 300, 600, 970 µg/mL
4 hr treatment (with activation): 75, 150, 300, 600, 970 µg/mL
20 hr treament (without activation): 75, 150, 300, 600, 970 µg/mL

Chromosome Aberration Assay:
Non-activated 4-hour exposure group:
At the highest test dose level evaluated microscopically for chromosome aberrations, 970 µg/mL, mitotic inhibition was 7%, relative to the vehicle control. The dose levels selected for analysis of chromosome aberrations were 300, 600, and 970 µg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) group was statistically significant (24.0%) (p ≤ 0.01, Fisher's Exact test).

S9-activated 4-hour exposure group:
At the highest test dose level evaluated microscopically for chromosome aberrations, 970 µg/mL, mitotic inhibition was 11%, relative to the vehicle control. The dose levels selected for analysis of chromosome aberrations were 300, 600, and 970 µg/mL. The percentage of cells with structural aberrations demonstrated a statistically significant increase (3.5%) relative to vehicle control at dose level 970 µg/mL (p ≤ 0.01, Fisher's Exact test). The Cochran- Armitage test was also positive for a dose response (p ≤ 0.05). The percentage of cells with numerical aberrations in the S9-activated 4-hour exposure group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) group was statistically significant (15.0%) (p ≤ 0.01, Fisher's Exact test).

Non-activated 20-hour exposure group:
At the highest test dose level evaluated microscopically for chromosome aberrations, 970 µg/mL, mitotic inhibition was 30%, relative to the vehicle control. The dose levels selected for analysis of chromosome aberrations were 300, 600, and 970 µg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) group was statistically significant (30.0%) (p ≤ 0.01, Fisher's Exact test).

The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.

Summary Table of Results:

Metabolic Activation	Test Article	Concentration µg/mL	Cytotoxicity^a (% of Control)	Aberrant Cells		Aberrations per Cell ^b,^d	Total Polyploid Cells (Mean %)^e
				Structural(Mean %)^b	Numerical(Mean %)^c
20-hr Continuous	Water	NA	NA	0.0	0.0	0.000±0.000	0.0
Treatment	R-50845	300	11	0.0	0.0	0.000±0.000	0.0
Without	R-50845	600	22	0.0	0.0	0.000±0.000	0.0
Activation	R-50845	970	30	0.5	0.0	0.005±0.071	0.0
	MMC	0.3	49	30.0**	0.0	0.330±0.533	0.0
4-hr Treatment	Water	NA	NA	0.0	0.0	0.000±0.000	0.0
With 16 hr Recovery	R-50845	300	1	0.5	0.0	0.005±0.071	0.0
Without	R-50845	600	-5	0.0	0.0	0.000±0.000	0.0
Activation	R-50845	970	7	0.5	0.0	0.005±0.071	0.0
	MMC	0.6	50	24.0**	0.0	0.260±0.485	0.0
4-hr Treatment	Water	NA	NA	0.0	0.0	0.000±0.000	0.0
With 16 hr Recovery	R-50845	300	4	0.0	0.0	0.000±0.000	0.0
With	R-50845	600	11	1.5	0.0	0.015±0.122	0.0
Activation	R-50845	970	11	3.5**	0.0	0.045±0.271	0.0
	CP	5	48	15.0**	0.0	0.150±0.359	0.0

MMC:Mitomycin C; CP:Cyclophosphamide; NA:NotApplicable; Fisher’s Exact Test:* p ≤0.05; **p ≤0 .01.

a.        Based on mitotic inhibition relative to solvent control.

b.       Does not include cells with only gaps.

c.        Includes polyploid and endoreduplicated cells.

d.       Severely damaged cells counted as 10 aberrations.

e.        Does not include endoreduplicated cell.

Conclusions:

The positive and vehicle controls fulfilled the requirements for a valid test.

Under the conditions of the assay described in this report, the test article was concluded to be negative in non-activated and positive in the S9 activated treatment for structural aberrations. The test article was concluded to be negative for numerical aberrations in both non-activated and S9-activated in the in vitro chromosome aberration assay in HPBL under the conditions, and according to the criteria of the study protocol.

Executive summary:

The test article was tested in the chromosome aberration assay using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system. A preliminary toxicity test was performed to establish the dose range for testing in the cytogenetic test. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article.

Water was used as the vehicle based on the solubility information provided by the Sponsor and compatibility with the target cells. Cyclophosphamide and mitomycin C were evaluated as the concurrent positive controls for treatments with and without S9, respectively.

In the preliminary toxicity assay, the doses tested ranged from 0.097 to 970 µg/mL (10 mM). Human peripheral blood lymphocytes were treated in the absence and presence of an Aroclor-induced S9 activation system for 4 hours, and continuously for 20 hours in the absence of S9 activation. Substantial toxicity (at least 50% reduction in mitotic index relative to the vehicle control) was not observed at any dose level in any of the three treatment groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 75 to 970 µg/mL for all three treatment conditions.

In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. In the absence of cytotoxicity or visible precipitate in the treatment medium, the highest dose evaluated for chromosome aberrations was the highest dose tested in the definitive assay (10 mM), which met the dose limit as recommended by testing guidelines for this assay. Two additional dose levels were included in the evaluation.

No statistically significant or dose-dependent increases in structural aberrations was observed in the non activated 4 and 20 hr treatment. A statistically significant and dose-dependent increases in structural aberrations was observed with S9 treatment (3.5% aberrant cells at 970

µg/mL; p ≤ 0.01; Fisher’s Exact and Cochran-Armitage tests). No statistically significant or dose-dependent increases were observed in the numerical aberrations (polyploidy and/or endoreduplicated cells) in any of the treatment condition.

All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases. Thus, all criteria for a valid study were met.

These results indicate the test article was negative in non-activated and positive in the S9 activated treatment for structural aberrations. The test item was negative for numerical aberrations in both non-activated and S9-activated in the in vitro chromosome aberration assay in HPBL under the conditions, and according to the criteria of the study protocol.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Genetic toxicity (in vivo mammalian erythrocyte micronucleus test) = Negative, OECD 474, BioReliance

Link to relevant study records

Reference

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

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

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.

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

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

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

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

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

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

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

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

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

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

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

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Definitive assay - vehicle (Group 1)

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.

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).

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the three genetic toxicity studies the substance does not meet the criteria for mutagenicity in accordance with Regulation (EC) No 1272/2008.