3-butoxypropylamine

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:

12 January 2018 to 09 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Updated guidance adopted 29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO/IEC 17025:2005 (ISO/IEC, 2005)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

3-butoxypropylamine

EC Number:

240-566-8

EC Name:

3-butoxypropylamine

Cas Number:

16499-88-0

Molecular formula:

C7H17NO

IUPAC Name:

3-butoxypropylamine

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PFW160573
- Expiration date of the lot/batch: 2018-09-26
- Purity test date: 2018-09-26

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature, protected from light
- Stability under test conditions: not been determined to cover the period of shipment and storage at the laboratory

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

(Hsd:SD)

Details on species / strain selection:

This species has been routinely used as an animal model of choice for the mammalian bone marrow erythrocyte micronucleus assay. This strain is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to the test substance.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS, Inc., Frederick, MD
- Age at study initiation: 6 weeks
- Weight at study initiation (Day 1): Females: 155.5 - 167.3 grams; Males: 200.5 - 202.6 grams
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: 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.
- Diet (e.g. ad libitum): rodent chow ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 10 changes of fresh HEPA-filtered air per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: Deionized water was used as the vehicle based on the solubility of the test article, and compatibility with the test system.
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared prior to dose administration as follows:
A suitably sized amber glass vial with a PTFE stir bar was calibrated to the target batch size. An appropriate amount of test substance was added to the vial. Approximately 70% of the total volume of deionized water was added to the test substance in the vial and stirred. The formulation was QS to the final volume with the vehicle and stirred magnetically until uniform.

All dose formulations were administered once at a volume of 10 mL/kg by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles).

Duration of treatment / exposure:

One single administration

Frequency of treatment:

One single administration

Post exposure period:

Sampling time: 24 (all dose levels) and 48h (control group and high dose level gorup) after a single administration of the test item the bone marrow cells were collected for micronuclei analysis

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control)
Sampling time: 24 and 48h after administration

Doses / concentrations 2

Dose / conc.:

42.5 mg/kg bw/day (nominal)

Remarks:

Group 2
Sampling time: 24h after administration

Doses / concentrations 3

Dose / conc.:

85 mg/kg bw/day (nominal)

Remarks:

Group 3
Sampling time: 24h after administration

Doses / concentrations 4

Dose / conc.:

170 mg/kg bw/day (nominal)

Remarks:

Group 4
Sampling time: 24 and 48h after administration

No. of animals per sex per dose:

5 animals
2 additional animals were treated in the high dose group for the 24h sampling time to cover in the event of mortality. Only 5 animals were euthanized at the 24 hour time point and additional animals were retained for possible use at the 48 hour time point. Only 5 animals were used for the 48 hour time point, any additional animals were euthanized without collection or further examination.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate
- Justification for choice of positive control(s): The positive control substances have been characterized as per the Certificate of Analysis on file with the testing facility. The stability of the positive control substance and its mixture was demonstrated by acceptable results that met the criteria for a valid test.
- Route of administration: oral gavage, once
- Doses / concentrations: dissolved in deionised water, administered at 40 mg/kg. The maximum dose evaluated for micronucleus induction was the MTD. Scoring positive control slides (fixed and unstained), generated from BioReliance Study No. AE93CB.125M012.BTL were included to verify scoring. These slides were generated from male rats treated once with cyclophosphamide monohydrate (CP) at 40, and the bone marrow harvested 24 hours after treatment

Examinations

Tissues and cell types examined:

Femoral bone marrow - 4000 polychromatic erythrocytes were analysed per animal for micronuclei.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In the DRF assay, 3 animals/sex (males and females) were exposed to 170 and 85 mg/kg of 3-Butoxypropylamine. Following the last observation, animals were euthanized by exposure to CO2 and discarded without further examination. No mortality occurred and no substantial differences in clinical observations were seen between the sexes, therefore only male animals were used in the definitive assay.

DETAILS OF SLIDE PREPARATION: Femoral bone marrow was collected at approximately 24 or 48 hours after the final dose, as indicated above. 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 2 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 four slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of two slides (including at least five positive control slides) was stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup and will be archived at report finalization. Stained slides will be discarded prior to report finalization. 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.

METHOD OF 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).
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.
4000 PCEs/animal were scored for the presence of micronuclei (MnPCEs). In addition, at least 500 total erythrocytes (PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity.

Evaluation criteria:

A test substance was considered to have induced a positive response if:
a) at least one of the test substance doses exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p≤ 0.01 and R²≥70%), and
c) results of the group mean or of the individual animals in at least one group were outside the 95% control limit of the historical negative control data.

A test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met and there was evidence that the bone marrow was exposed to the test substance (unless intravenous administration was used).

Statistics:

Statistical analysis was performed on the micronucleus frequency (%MnPCE) and %PCE using the animal as the unit. The mean and standard deviation of %MnPCE and %PCE were presented for each treatment group.
The use of parametric or non-parametric statistical methods in the evaluation of data was based on the variation between groups. The group variances for micronucleus frequency for the vehicle and test substance groups at the respective sampling time were compared using Levene’s test (significance level of p <= 0.05).
A linear regression analysis was conducted to assess dose responsiveness in the test substance treated groups (p<= 0.01 and R2≥70%).
A pair-wise comparison (Student’s T-test) was used to compare the positive control group to the concurrent vehicle control group.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: No mortality occurred at any dose level during the course of the dose range finding assay. No reductions in mean group body weights were seen in the test substance treated groups during the course of the study. Piloerection was observed in males and females at 85 mg/kg. Piloerection and hunched position was observed in males and females at 170 mg/kg. Based on sponsor-provided information indicating approximately half of the animals died after a single administration of 175 mg/kg and the results of the range finding assay, 170 mg/kg was determined to be the maximum tolerated dose and was used as the high dose level for the definitive assay.
- Rationale for exposure: maximum guideline-recommended dose
- 2 day observation period following administration


RESULTS OF DEFINITIVE STUDY
- Clinical signs: No mortality occurred at any dose level during the course of the definitive assay. No reductions in mean group body weights were seen in the test substance treated groups during the course of the study. Piloerection was observed at 170 mg/kg.
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): No reductions in the PCEs/EC ratio were observed in the test substance groups compared to the vehicle control group, indicating the test substance did not induce cytotoxicity.
- Genotoxicity reuslts and statistical evaluation: Group variances the mean of the micronucleus frequency in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data. No statistically significant increase in the incidence of MnPCEs was observed in the test substance treated groups relative to the vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).

The positive control, CP, induced a statistically significant increase in the incidence of MnPCEs (Student’s t-test, p ≤ 0.05).
The number of MnPCEs in the vehicle control groups did not exceed the historical control range.
Based upon this, all criteria for a valid test were met as specified in the protocol.

Any other information on results incl. tables

Dose Formulation Analysis

Dose formulations were sent to the analytical chemistry laboratory at BioReliance. A copy of the analytical report is included in Appendix IV. The results of the analysis indicate that the actual mean concentrations of the analyzed formulation samples, 4.25, 8.5 and 17 mg/mL, were between 100.4-103.2% of target with S/L ratios of > 0.925. This indicates that the formulations were accurately prepared. No test substance was detected in the vehicle control sample.

Additionally, 3-Butoxypropylamine in deionized water, at a concentration of 17.1 mg/mL, was stable at room temperature for at least 3.5 hours.

The 3-hour stability sample for the low dose (4.25 mg/mL) did not meet the protocol acceptance criterion of 90-110% of the concentration determined at T=0 (actual: 87.5% of target). The animals at this dose level were dosed between 0926 and 0928 the same morning that the samples were analyzed. All formulation samples were delivered to the analytical chemistry laboratory at 0928, and the dilutions for the T=0 analysis were started at 0945. Based on this, and the T=0 dose formulation analysis samples being within range (100.4% of target), it is assumed that the animals in this group were dosed with the dose level intended.

Summary of Bone Marrow Micronucleus Analysis

Treatment	Gender	(Hrs)	Animals	(Mean +/- SD)			(%)	(Mean +/- SD)			MnPCE/PCE Scored
Vehicle-Deionized water 0 mg/kg	M	24	5	54.4	±	7.8	---	0.07	±	0.03	14	/20000
3-Butoxypropylamine 42.5 mg/kg	M	24	5	57.4	±	10.2	6	0.07	±	0.04	14	/20000
85 mg/kg	M	24	5	56.6	±	10	4		±	0.04	11	/20000
170 mg/kg	M	24	5	57.2	±	11.1	5	0.09	±	0.01	18	/20000
Vehicle 0 mg/kg	M	48	5	52.9	±	9.3	---	0.09	±	0.02	17	/20000
3-Butoxypropylamine 170 mg/kg	M	48	5	56.5	±	6.3	7	0.09	±	0.03	18	/20000
CP 40 mg/kg	M	24	5	27.4	±	5.5**	-50	3.36	±	0.42**	671	/20000

*p < 0.05 or **p < 0.01, One-Way ANOVA with Post-Hoc Dunnett's Test or T-Test

24 Hrs MnPCE Male GLM P-value = 0.405, R-sqr = 16.20%

PCE – Polychromatic Erythrocytes; MnPCE – Micronucleated Polychromatic Erythrocytes

Rat Micronucleus Test Historical Control Data

2012-2015Historical Vehicle Control in Male Rats^1
Individual Animals				Studies
PCE%		MN%		PCE%		MN%
N	1047		1047		209		209
Mean^3	52.4		0.06		52.4		0.06
SD	5.9		0.06		4.5		0.04
95% UCL	64.1		0.18		61.4		0.15
95% LCL	40.7		0.00		43.4		0.00
Max^4	73.4		0.30		65.7		0.23
Min^4	22.2		0.00		41.0		0.00
Historical Positive Control in Male Rats^2
Individual Animals				Studies
PCE%		MN%		PCE%		MN%
N	749		759		151		153
Mean^3	47.7		2.51		47.7		2.51
SD	8.6		1.03		7.7		0.91
95% UCL	64.9		4.57		63.2		4.33
95% LCL	30.5		0.46		32.3		0.68
Max^4	72.7		6.65		61.7		5.15
Min^4	12.0		0.18		25.3		0.30

 1Since no appreciable differences in the induction of MPCEs by different vehicles and solvents (test substance carriers) and different routes of administration were observed, this table contains data from carriers and routes of administration widely used during the conduct of contract studies at BioReliance.

Vehicles: water, water soluble vehicles (methylcellulose, carboxymethylcellulose, dextrose), saline, corn oil and other vehicles.

Routes of administration: intraperitoneal (IP), intravenous (IV), oral gavage (PO), subcutaneous (SC).

Bone marrow collection time: 24 and 48 hours post-dose.

2Positive control substance: Cyclophosphamide monohydrate (CP); Doses: 40 mg/kg; Route of administration: PO.

3Average of the PCE ratio observed out of 500 or 1000 erythrocytes scored per animal for the total number of animals used; average of the number of MPCE per 2000 or 4000 PCE for the total number of animals used; average of number of MPCE/per group (containing 5-7 animals per group) for total number of groups used.

4Minimum and maximum range of PCE ratio observed out of 500 or 1000 erythrocytes scored per animal, the minimum and maximum range of MPCE observed out of 2000 or 4000 PCE for the total number of animals used and the minimum and maximum range of MPCE observed out of 10000 to 24000 PCE for the total number of groups used.

Formula: 95% control limit ranges = mean ± 2 x standard deviation

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the oral administration of 3-Butoxypropylamine at doses up to and including a dose of 170 mg/kg was concluded to be negative for clastogenic activity and/or disruption of the mitotic apparatus in the Micronucleus assay.