bis(diethylamino)-N,N-diethylmethaniminium chloride

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 474 (1998); EPA OPPTS 870.5395 (1998); GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

The ICR mice were obtained from Harlan Sprague Dawley, Inc., Frederick, MD and were approximately 6-8 weeks of age at study initiation, weighing 26.6 32.5 g (males) and 24.3-30.2 g (females). Up to five mice of the same sex were group housed in polycarbonate cages with heat-treated hardwood chips for bedding. The controlled environment parameters were 72 ± 3°F, 50 ± 20% relative humidity and a 12-hour light/dark cycle. The mice had free access to certified rodent chow and water.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Water (CAS No. 7732-18-5); from Invitrogen Corporation

Details on exposure:

In the pilot assay, five male and five female mice were exposed to the test substance at a dose of 2000 mg/kg and two male mice each to 1, 10, 100, or 1000 mg/kg. Test substance dosing formulations were administered at a volume of 20 mL/kg by a single IP injection. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration and 1 and 3 days after dose administration.

For the toxicity assay, all mice were weighed immediately prior to dose administration and the dose volume was based on individual body weight. In the toxicity study, animals (5 animals/sex/group) were dosed at 8.8, 20, 40, or 60 mg/kg body weight. Test substance dosing formulations were administered at a volume of 20 mL/kg by a single IP injection. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration and 1 and 3 days after dose administration.

For the definitive micronucleus assay, seven groups, each containing 5 male and 5 female ICR mice. Animals in five of these groups were treated either with the controls (negative or positive) or with test substance at a dose of 2.5, 5.0 or 10 mg/kg and were euthanized 24 hours after treatment. Animals in the other two groups were treated either with the negative control or test substance at a dose of 10 mg/kg and were euthanized 48 hours after treatment. Additional replacement animals (5 animals/sex) were included in the high dose group, 10 mg/kg, to ensure that the availability of 5 animals/sex for micronucleus analysis. The test substance vehicle mixture, the vehicle alone or the positive control was administered by a single IP injection at a dose volume of 20 mL/kg. All mice in the experimental and control groups were weighed immediately before dose administration and the dose volume was based on individual body weight. Mice were observed after dose administration for clinical signs of toxicity.

Frequency of treatment:

Single treatment

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1, 10, 100, 1000 or 2000 mg/kg body weight
Basis:
nominal conc.
Pilot assay

Doses / concentrations 2

Remarks:

Doses / Concentrations:
8.8, 20, 40, or 60 mg/kg body weight
Basis:
nominal conc.
Toxicity Assay

Doses / concentrations 3

Remarks:

Doses / Concentrations:
2.5, 5.0 or 10 mg/kg body weigh
Basis:
nominal conc.
Definitive Assay

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes, Cyclophosphamide monohydrate (CP, CAS No. 6055-19-2); 2.5 mg/mL

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

At the scheduled sacrifice times, five mice per sex per dose were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes, and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May Gruenwald Giemsa and permanently mounted.

Bone marrow cells [polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)], were analyzed for the presence of micronuclei. Polychromatic erythrocytes are young, immature red blood cells that stain bluish while normochromatic erythrocytes or normocytes are mature red blood cells that stain pink. Micronuclei are round, darkly-staining nuclear fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei can occur in both PCEs (MPCEs) and NCEs (MNCEs).
To control for bias, slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification (10 x 40), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (10 x 100), 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes

Evaluation criteria:

To quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and dose group.
As a guide to interpretation of the data, test substance was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p less than or equal to 0.05, Kastenbaum Bowman Tables) at any sampling time. However, values that were statistically significant but did not exceed the range of historical negative or vehicle controls were judged as not biologically significant. The test substance was judged negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response were observed at any sampling time.

Statistics:

The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and dose group. Statistical significance was determined using the Kastenbaum Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the pilot study, the test substance was administered to male and female mice at 1, 10, 100, 1000 or 2000 mg/kg. Mortality was observed in 2/2 male mice at 100 and 1000 mg/kg and in 5/5 male and 5/5 female mice at 2000 mg/kg. Clinical signs following dose administration included: slight ataxia in males at 10 mg/kg and convulsions in males at 1000 mg/kg and in males and females at 2000 mg/kg.

In the toxicity study, the test substance was administered to male and female mice (5/sex/group) at 8.8, 20, 40, or 60 mg/kg. Two females at 20 mg/kg, four males and all females at 40 mg/kg, and all animals at 60 mg/kg were found dead following dose administration. Due to mortality immediately after dose administration, clinical signs were not observed in 4/5 males and all females at 40 mg/kg and in all males and females at 60 mg/kg. All animals at 8.8 mg/kg, all surviving animals at 20 mg/kg and one male mouse at 40 mg/kg appeared normal during the study period. Based upon these results, the high dose for the micronucleus test was set at 10 mg/kg, which was estimated to be the maximum tolerated dose.

In the definitive micronucleus study, the test substance was administered to male and female mice (5/sex/group) at 2.5, 5.0 or 10 mg/kg. No mortality occurred at any dose during the course of the micronucleus study. Clinical signs following dose administration included: piloerection in males at 2.5 mg/kg and in males and females at 5.0 and 10 mg/kg. All other animals treated with the test or control articles appeared normal during the course of the study.

Bone marrow cells (polychromatic erythrocytes, PCEs and normochromatic erythrocytes, NCEs), collected 24 and 48 hours after treatment were examined microscopically for presence of micronuclei (MPCEs or MNCEs). Reductions, up to 21%, in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. These reductions suggest bioavailability of the test article to the bone marrow target. The number of micronucleated polychromatic erythrocytes per 10,000 polychromatic erythrocytes in the test substance-treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose or bone marrow collection time (p greater than 0.05, Kastenbaum-Bowman Tables). In addition, no appreciable increase in the number of MNCEs in the field of 2000 PCEs per animal was found, indicating that an optimal differential staining was achieved.

In this study, all criteria for a valid test were met as specified in the protocol. CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p less than 0.05, Kastenbaum-Bowman Tables). The negative and positive controls were consistent with the historical control data, indicating that there was no problem with the test system or the quality of the test.

Any other information on results incl. tables

Summary of Bone Marrow Micronucleus Analysis Following a Single Dose of the Test Substance in ICR Mice

Treatment (20 mL/kg)	Sex	Time (hr)	Number of Mice	PCE/Total             Erythrocytes        (Mean +/- SD)	Change from Control (%)	Micronucleated Polychromatic Erythrocytes
						Number per 1000 PCEs (Mean +/- SD)	Number per PCEs Scored*
Water	M	24	5	0.464 ± 0.02	---	0.5 ± 0.35	5 / 10000
	F	24	5	0.480 ± 0.04	---	0.3 ± 0.27	3 / 10000
Test Substance
2.5 mg/kg	M	24	5	0.502 ± 0.03	8	0.5 ± 0.00	5 / 10000
	F	24	5	0.476 ± 0.06	-1	0.2 ± 0.27	2 / 10000
5.0 mg/kg	M	24	5	0.478 ± 0.04	3	0.4 ± 0.22	4 / 10000
	F	24	5	0.424 ± 0.08	-12	0.4 ± 0.22	4 / 10000
10 mg/kg	M	24	5	0.429 ± 0.06	-8	0.3 ± 0.27	3 / 10000
	F	24	5	0.379 ± 0.05	-21	0.4 ± 0.22	4 / 10000
CP
50 mg/kg	M	24	5	0.395 ± 0.06	-15	13.1 ± 3.80	*131 / 10000
	F	24	5	0.368 ± 0.08	-23	12.8 ± 3.05	*128 / 10000
Water	M	48	5	0.501 ± 0.06	---	0.3 ± 0.27	3 / 10000
	F	48	5	0.491 ± 0.04	---	0.7 ± 0.45	7 / 10000
Test Substance
10 mg/kg	M	48	5	0.478 ± 0.04	-5	0.4 ± 0.42	4 / 10000
	F	48	5	0.487 ± 0.06	-1	0.1 ± 0.22	1 / 10000
*Statistically significant, p less than or equal to 0.05 (Kastenbaum‑Bowman Tables)

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 24 Hours Following a Single Dose of Test Substance in ICR Mice

Treatment (20 mL/kg)	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Water	M	101	0.435	2 / 2000
		102	0.470	1 / 2000
		103	0.465	0 / 2000
		104	0.485	1 / 2000
		105	0.466	1 / 2000
	F	106	0.464	0 / 2000
		107	0.441	1 / 2000
		108	0.453	1 / 2000
		109	0.518	0 / 2000
		110	0.523	1 / 2000
Test Substance
2.5 mg/kg	M	111	0.492	1 / 2000
		112	0.555	1 / 2000
		113	0.512	1 / 2000
		114	0.487	1 / 2000
		115	0.464	1 / 2000
	F	116	0.438	0 / 2000
		117	0.581	0 / 2000
		118	0.461	1 / 2000
		119	0.453	0 / 2000
		120	0.446	1 / 2000
5.0 mg/kg	M	121	0.469	1 / 2000
		122	0.483	1 / 2000
		123	0.415	1 / 2000
		124	0.503	1 / 2000
		125	0.518	0 / 2000
	F	126	0.434	1 / 2000
		127	0.300	1 / 2000
		128	0.523	1 / 2000
		129	0.440	0 / 2000
		130	0.421	1 / 2000
10 mg/kg	M	131	0.521	1 / 2000
		132	0.427	1 / 2000
		133	0.415	1 / 2000
		134	0.355	0 / 2000
		135	0.425	0 / 2000
	F	136	0.335	1 / 2000
		137	0.418	1 / 2000
		138	0.319	1 / 2000
		139	0.416	0 / 2000
		140	0.408	1 / 2000
CP 50 mg/kg	M	141	0.352	35 / 2000
		142	0.372	25 / 2000
		143	0.472	20 / 2000
		144	0.447	18 / 2000
		145	0.330	33 / 2000
	F	146	0.327	24 / 2000
		147	0.496	35 / 2000
		148	0.385	20 / 2000
		149	0.310	21 / 2000
		150	0.321	28 / 2000

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 48 Hours Following a Single Dose of Test Substance in ICR Mice

Treatment (20 mL/kg)	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Water	M	151	0.563	1 / 2000
		152	0.505	1 / 2000
		153	0.419	0 / 2000
		154	0.472	1 / 2000
		155	0.544	0 / 2000
	F	156	0.538	2 / 2000
		157	0.524	2 / 2000
		158	0.488	0 / 2000
		159	0.455	2 / 2000
		160	0.451	1 / 2000
Test Substance
10 mg/kg	M	161	0.481	1 / 2000
		162	0.429	1 / 2000
		163	0.466	0 / 2000
		164	0.531	2 / 2000
		165	0.485	0 / 2000
	F	166	0.447	0 / 2000
		167	0.425	0 / 2000
		168	0.585	0 / 2000
		169	0.494	0 / 2000
		170	0.486	1 / 2000

Applicant's summary and conclusion

Conclusions:

Under the conditions of the assay described in this report, a single intraperitoneal administration of the test substance at doses up to 10 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the test substance was concluded to be negative in the micronucleus test using male and female ICR mice.