3-butoxypropylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three reliable, key in vitro genetic toxicity studies are available.

Ames: The test item did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

HPRT: The test substance was concluded to be negative for the induction of forward mutations at the HPRT locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation assay.

CAT: The test item was concluded to be positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in the non-activated test system; and negative for induction of structural and numerical chromosome aberrations in the S9-activated test system.

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

Study period:

13 February 2014 to 13 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 8291-11-20
- Expiration date of the lot/batch: 2014-07-13
- Purity test date: 2012-07-13

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2 to 8ºC, protected from light

Target gene:

Histidine locus (histidine-dependent Salmonella strains) and tryptophan locus (tryptophan-dependent E. coli strain)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Initial Toxicity-Mutation Assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate with and without metabolic activation.
Confirmatory Mutagenicity Assay: 50, 150, 500, 1500 and 5000 μg per plate with and without metabolic activation.

Since the test item formed a clear solution in sterile water for injection at approx. 50 mg/mL, 5000 µg/plate was selected as the maximum concentration in the initial toxicity-mutation assay.
Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Sterile water for injection was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article formed a clear solution in sterile water for injection at approximately 50 mg/mL, the maximum concentration tested in the solubility test conducted at the laboratory

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9: 1.0 μg/plate for TA98, TA1535 ; 2.0 μg/plate for TA100, TA1537; 15 μg/plate for WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9: 1.0 μg/plate for TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9: 1.0 μg/plate for TA100, TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9: 75 μg/plate for TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9: 1.0 μg/plate for WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water. Bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).
Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain and activation. One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 100 μL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

DURATION
- Exposure duration: 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium) or Tryptophan (E. coli)

NUMBER OF REPLICATIONS:
Initial toxicity-mutation assay: duplicate
Confirmatory mutagenicity assay: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the growth of the bacterial background lawn and/ reduction in the number of revertants

Evaluation criteria:

For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article.
Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative, if it was neither positive nor equivocal.

Statistics:

No formal hypothesis testing was done.
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Soluble at 50 mg/mL
- Precipitation: no precipitation has been observed
- Other: sterility results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions or the S9 and Sham mixes.

RANGE-FINDING/SCREENING STUDIES:
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. Vehicle control, positive controls and eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: all positive controls exhibited at least 3-fold increases in the number of revertants
- Negative (solvent/vehicle) historical control data: the number of revertants was within the characteristic ranges for all vehicle controls
All criteria for a valid study were met as described in the protocol

Conclusions:

Interpretation of results: negative with and without metabolic activation
The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test item did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 2017 to 27 October 2017

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)

Version / remarks:

Updated and adopted 26 September 2014

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name: 3-Butoxypropylamine
- Description: clear, colorless liquid
- Source and lot/batch No.of test material: PFW160573
- Expiration date of the lot/batch: 2018-09-26
- Purity: 100%
- Purity test date: 2016-09-30

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- STORAGE CONDITIONS: Room Temperature, protected from light
- Analyses to determine the concentration, uniformity and stability of the test substance dose formulations were not performed.

OTHER SPECIFICS:
Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.
Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle and that of the highest dose was measured.
The pH of the highest dose of dosing solution in the treatment medium was measured using test tape. When necessary, in order to maintain neutral pH in the treatment medium, pH was adjusted using 1N HCl.

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: American Type Culture Collection, Manassas, VA
- Suitability of cells: The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens
- Normal cell cycle time (negative control): 10-14 hours

For cell lines:
- Absence of Mycoplasma contamination: The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination.
- Number of passages if applicable: 15 max
- Methods for maintenance in cell culture: not indicated
- Cell cycle length, doubling time or proliferation index : 10-14h
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: yes, in order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15.


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16-24 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test
0, 0.131, 0.393, 1.31, 3.93, 13.1, 39.3, 131, 393 and 1310 µg/mL with and without S9-mix (4-hour treatment) and without S9-mix (20-hour treatment);

Chromosome Aberration Assay
Treatment time: 4 hours with recovery time 16 hours: 0, 10, 30, 100, 325, 650, 1310 µg/mL without S-9 mix;
Treatment time: 20 hours with recovery time 0 hours: 0, 5, 10, 25, 30, 35, 40, 45, 50 µg/mL without S-9 mix;
Treatment time : 4 hours with recovery time 16 hours: 0, 10, 30, 100, 325, 650, 1310 µg/mL with S-9 mix;

In the preliminary toxicity assay, the doses tested ranged from 0.131 to 1310 µg/mL (=10 mM), which was the limit dose for this assay.
In the definitive chromosome aberration assay, the doses chosen for the chromosome aberration assay ranged from 10 to 1310 µg/mL for the non activated and S9-activated 4-hour exposure groups and from 5 to 50 µg/mL for the non activated 20-hour exposure group, based on the absence of cytotoxicity or visible precipitate in the treatment medium in the preliminary toxicity test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9-mix; 0.1 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9-mix; 5 µg/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 5 x 10^5 cells/culture
- Test substance added in medium
Vehicle (Non-activated): 4.5 mL culture medium + 500 µL vehicle
Vehicle (S9-activated): 3.5 mL culture medium + 1 mL S9-mix + 500µL vehicle
Test Substance (Non-activated): 4.5 mL culture medium + 500 µL test item dosing solution
Test Substance (S9-activated): 3.5 mL culture medium + 1 mL S9-mix + 500 µL test item dosing solution
Positive Control (Non-activated) : 5 mL culture medium + 50 µL positive control dosing solution
Positive Control (S9-activated): 4 mL culture medium + 1 mL S9-mix + 50 µL positive control dosing solution

After the 4-hour treatment period in the non-activated and the S9-activated studies, the treatment medium was aspirated, the cells washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium and returned to the incubator under standard conditions. For the definitive assay only, two hours prior to cell harvest, Colcemid was added to all cultures at a final concentration of 0.1 µg/mL.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 and 20 hours without S9 and for 4 hours with S9
- Harvest time after the end of treatment (sampling/recovery times): 20 hours (+- 30 minutes)
- Spindle inhibitor: Two hours prior to cell harvest, Colcemid was added to all cultures at a final concentration of 0.1 µg/mL
- Methods of slide preparation and staining technique used including the stain used: For the definitive assay only, 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 to 8°C. To prepare slides, the cells were collected by centrifugation and 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, treatment condition, harvest date, activation system, test phase, and replicate tube design.
- Number of cells spread and analysed per concentration: A minimum of 300 metaphase spreads containing 20 ± 2 centromeres from each dose (150 per duplicate treatment) were examined and scored for chromatid-type and chromosome-type aberrations. 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 150 cells are scored.
- Criteria for scoring chromosome aberrations:
Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded. The XY vernier for each cell with a structural aberration was recorded. The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated for 150 cells per culture (a total of 300 per dose level).
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. Chromatid and isochromatid gaps were presented in the data but were not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative increase in cell count (RICC)
- Any supplementary information relevant to cytotoxicity: Just prior to harvest, the cell cultures was visually inspected for the degree of monolayer confluency relative to the vehicle control. The cells were trypsinized and counted and the cell viability was assessed using trypan blue dye exclusion. The cell count was determined from a minimum of two cultures to determine the number of cells being treated (baseline). The data were presented as cell growth inhibition in the treatment group compared to vehicle control.
The cell counts and percent viability were used to determine cell growth inhibition (CGI) relative to the vehicle control (% cytotoxicity = 100 – RICC).

Evaluation criteria:

The test substance was considered to have induced a positive response if
• at least one of the test concentrations exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase is concentration-related (p ≤ 0.05), and
• results are outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at doses ≥ 45 μg/mL in the non-activated 20-hour exposure group.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Preliminary Toxicity Assay
The test substance was soluble in the treatment medium at all doses tested at the beginning and conclusion of the treatment period.
The osmolality in treatment medium was measured as follows:
- 278 mmol/kg in the vehicle
- 291 mmol/kg at the highest dose tested (1310 µg/ml)
The osmolality of the test substance dose in treatment medium was considered acceptable (<120% of vehicle).
55 µL of 1N HCl were added to the highest dose tested (1310 µg/mL) to adapt the pH from 9.0 to 7.0

Chromosome Aberration Assay
The test substance was soluble in the treatment medium at all doses tested at the beginning and conclusion of the treatment period. The initial pH of the highest dose of test substance in treatment medium was 9.0. T
he pH was adjusted using 1N HCl as follows:
- 55 µL of 1N HCl were added to the highest dose tested (1310 µg/mL) to adapt the pH from 9.0 to 7.0
- 40 µL of 1N HCl were added to one of the intermediate doses tested (650 µg/mL) to adapt the pH from 8.0 to 7.0

RANGE-FINDING/SCREENING STUDIES:
CHO cells were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures.
Cytotoxicity (>= 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non activated and S9-activated 4-hour exposure groups. Cytotoxicity was observed at doses >= 39.3 µg/mL in the non-activated 20-hour exposure group. Based upon the results of the preliminary toxicity assay, the highest dose levels selected for the chromosome aberration assay were 1310 and 50 µg/mL for the 4-hour and 20-hour exposure groups, respectively.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The results for the positive and vehicle controls indicate that all criteria for a valid assay were met
- Cytoxicity:
Mitotic inhibition was 26% in the non-activated 4-hour exposure group, 30% in the non-activated 20-hour exposure group and 6% in the activated 4-hour exposure group

In the non-activated and S9-activated 4-hour exposure groups, no significant or dose-dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).
In the non-activated 20-hour exposure group, statistically significant and dose-dependent increases in structural aberrations (5.0% and 7.3%) were observed at doses 25 and 50 μg/mL (p ≤ 0.01; Fisher’s Exact test and p ≤ 0.05; Cochran-Armitage test). No significant or dose-dependent increases in numerical aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).
In order to confirm the positive response observed in the non-activated 20-hour exposure group, 4 additional dose levels of 30, 35, 40, and 45 μg/mL were included in the microscopic evaluation. Statistically significant and dose-dependent increases in structural aberrations (5.0%, 6.0%, 6.3%, and 4.7%) were observed at doses 30, 35, 40, and 45 μg/mL, respectively (p ≤ 0.01; Fisher’s Exact test and p ≤ 0.05; Cochran-Armitage test). No significant or dose-dependent increases in numerical aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC and mitotic index relative to solvent control.

Summary of the results

treatment (µg/mL)	S9 activation	treatment time	mean mitotic index	cells scored		aberrations per cell		cells with aberrations
				numerical	structural	mean	+/-sd	numerical (%)	structural (%)
water	-S9	4	14.9	300	300	0.020	+/-0.140	3.7	2.0
TS 325	-S9	4	14.4	300	300	0.013	+/-0.115	4.0	1.3
TS 650	-S9	4	14.2	300	300	0.017	+/-0.128	3.0	1.7
TS 1310	-S9	4	11.1	300	300	0.020	+/-0.140	6.3	2.0
water	+S9	4	15.1	300	300	0.023	+/-0.172	7.0	2.0
TS 325	+S9	4	13.0	300	300	0.027	+/-0.161	7.0	2.7
TS 650	+S9	4	14.4	300	300	0.020	+/-0.140	7.7	2.0
TS 1310	+S9	4	14.2	300	300	0.020	+/-0.140	7.3	2.0
CP 5	+S9	4	8.3	300	150	0.253	+/-0.507	1.7	22.0**
water	-S9	20	14.3	300	300	0.010	+/-0.100	2.7	1.0
TS 5	-S9	20	13.0	300	300	0.013	+/-0.115	2.3	1.3
TS 25	-S9	20	11.6	300	300	0.050	+/-0.218	2.7	5.0**
TS 30	-S9	20	13.3	300	300	0.050	+/-0.218	1.3	5.0**
TS 35	-S9	20	12.5	300	300	0.073	+/-0.308	2.0	6.0**
TS 40	-S9	20	11.1	300	300	0.077	+/-0.313	1.7	6.3**
TS 45	-S9	20	10.7	300	300	0.053	+/-0.266	1.3	4.7**
TS 50	-S9	20	10.0	300	300	0.087	+/-0.336	3.0	7.3**
MMC 0.1	-S9	20	8.8	300	150	0.140	+/-0.367	2.3	13.3**

 TS: test substance

Treatment: cells from all treatment conditions were harvested 20 hours after the initiation of the treatments

Aberrations per cell: severely damaged cells were counted as 10 aberrations

Percent aberrant cells: *p<=0.05; **p<=0.01; using Fischer's Exact test

HISTORICAL CONTROL VALUES STRUCTURAL ABERRATIONS

2013-2015

 

NON-ACTIVATED TEST SYSTEM

 

Historical Values	Structural Aberration (%)
	Negative Control^1		Positive Controls^2
	4-hour	20-hour	4-hour	20-hour
Mean	0.74	0.64	21.07	20.87
Standard Deviation	±0.83	±0.78	±5.41	±5.21
95% Control Limits	0.00 - 2.41	0.00 - 2.21	10.25 - 31.88	10.45 - 31.29
Range^4	0.00 - 3.00	0.00 - 3.50	7.50 - 33.00	7.00 - 34.00

 

 

S9-ACTIVATED TEST SYSTEM

 

Historical Values	Structural Aberration (%)
	Negative Control^1	Positive Control^3
Mean	1.09	26.60
Standard Deviation	±1.39	±7.66
95% Control Limits	0.00 - 3.88	11.28 - 41.92
Range^4	0.00 - 9.50	12.50 - 52.00

 

1.

 Solvents include water, saline, DMSO, ethanol, acetone, and other non-standard and Sponsor supplied vehicles.

2.

 Positive control for non-activated studies, Mitomycin C (MMC).

3.

 Positive control for S9-activated studies, Cyclophosphamide (CP).

4.

 Range from minimum to maximum.

 

HISTORICAL CONTROL VALUES NUMERICAL ABERRATIONS

2013-2015

 

NON-ACTIVATED TEST SYSTEM

 

Historical Values	Numerical Aberration (%)
	Negative Control^1
	4-hour	20-hour
Mean	1.56	1.55
Standard Deviation	±1.08	±1.24
95% Control Limits	0.00 - 3.72	0.00 - 4.03
Range^2	0.00 -5.00	0.00 - 5.50

 

 

S9-ACTIVATED TEST SYSTEM

 

Historical Values	Numerical Aberration (%)
	Negative Control^1
Mean	2.68
Standard Deviation	±1.84
95% Control Limits	0.00 -6.35
Range^2	0.00 -10.00

 

1.

 Solvents include water, saline, DMSO, ethanol, acetone, and other non-standard and Sponsor supplied vehicles.

2.

 Range from minimum to maximum.

 

Conclusions:

Under the conditions of the assay described in this report, 3-Butoxypropylamine was concluded to be positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in the non-activated test system. 3-Butoxypropylamine was concluded to be negative for the induction of structural and numerical chromosome aberrations in the S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells. Hence, based on the results of this study 3-Butoxypropylamine is considered to be clastogenic in the non-activated test system in the in vitro mammalian chromosome aberration test using CHO cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June 2017 to 06 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline 476, updated and adopted 29 July 2016 and ISO/IEC 17025:2005 (ISO/IEC, 2005)

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

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: 26 September 2018
- Purity test date: 30 September 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Stability under test conditions: not determined to cover the period of shipment and storage at the testing facility
- Solubility of the test substance in the solvent/vehicle: The test substance formed a clear solution in water at a concentration of approximately 50 mg/mL in the solubility test

Target gene:

The purpose of this study was to evaluate a test article for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. Abraham W. Hsie, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN
- Suitability of cells: recommended by Guideline
- Modal number of chromosomes: 20

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
CHO cells were maintained in Ham's F12 medium supplemented with 3 mM L-glutamine and 5% (v/v) heat-inactivated and dialyzed fetal bovine serum (Complete Ham’s F12) under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air). All media contained antimycotics and antibiotics.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity assay: 2.56, 5.13, 10.3, 20.5, 41.0, 82.0, 164, 328, 656 and 1312 µg/mL.
Main mutation assays (first, second and final): 82, 164, 328, 656 and 1312 µg/mL with and without S9

The maximum concentration evaluated was based on solubility limitations of the test substance in the vehicle. Based upon these results, the maximum concentration chosen for the definitive mutagenicity assays (first, second and final) was 1312 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water for test substance. Positive Controls: Ethylmethanesulfonate (EMS) and Benzo(a)pyrene [B(a)P] was also diluted in DMSO.
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9-mix; final concentration 0.200 µL/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9-mix; final concentration 4.00 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Preliminary toxicity test:
Cells were plated (on Day -1) in 25-cm2 cultures at a density of ~1 x 10^6 in 5 mL Complete Ham’s F12. Following an overnight incubation under standard conditions, the cultures were washed twice (on Day 0) with Hank’s Balanced Salt Solution (HBSS) and re-fed with 4.5 mL treatment medium, or 3.5 mL treatment medium plus 1.0 mL S9 mix, as appropriate. Following addition of the test or control substance dose formulations (500 μL) to the flasks, the cultures were incubated under standard conditions for 5 ± 0.5 hours.
Main Mutation assays:
Cells were plated (on Day -1) in 75-cm2 cultures at a density of ~5 x 10^6 in 10 mL Complete Ham’s F12.
Following an overnight incubation (on Day 0) at standard conditions, the cultures were washed twice with HBSS and re-fed with 10 mL treatment medium, or 8 mL treatment medium plus 2 mL S9 mix (adjusted for the test substance dose volume if >1%, v/v), as appropriate. Following addition of the test or control substance formulations (1.2 mL for the first definitive mutagenicity assay and 1.1 mL for the second and final mutagenicity assay) to the flasks, the cultures were incubated under standard conditions for 5 ± 0.5 hours (positive control substances were prepared in DMSO and added to the flasks using a 1% dose volume).

DURATION
- Exposure duration: 5 +/- 0.5 hours
- Expression time (cells in growth medium): 7 to 8 days
- Selection time (if incubation with a selection agent): 7 to 10 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: single cultures in the preliminary toxicity assay; duplicate cultures in the mutagenicity assays

NUMBER OF CELLS EVALUATED: 2.4 x 10^6 cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: adjusted relative survival

Evaluation criteria:

The test substance was considered to have produced a positive response if it induced a dose-dependent increase in mutation frequency and an increase exceeding 95% historical vehicle control limits in at least one test dose level(s) as compared with concurrent vehicle control (p<0.01). If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the result were considered equivocal. If none of these criteria were 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.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Statistics:

Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The average adjusted relative survival was 103.7% at a concentration of 1312 µg/mL with S9. The average adjusted relative survival was 83.83% at a concentration of 1312 µg/mL without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
Preliminary toxicity assay: The pH of the treatment medium was measured and adjusted at concentrations ≥82.0 µg/mL using 1N hydrochloric acid (HCl; CAS No. 7647-01-0, Lot No. RNBD4214, Exp. Date: 31 Mar 2018). No pH adjustment was necessary to maintain neutral pH in the treatment medium at the remaining concentrations tested.
Definitive mutagenicity assays: The pH of the cultures was adjusted at concentrations ≥82 µg/mL with and without S9 to maintain neutral pH.
- Effects of osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%
- Precipitation: No visible precipitate was observed at the beginning or end of treatment

RANGE-FINDING/SCREENING STUDIES:
Cells were treated with 10 test substance concentrations, as well as the vehicle control, in the presence and absence of S9 using single cultures. The maximum concentration evaluated was selected based on test substance solubility limitations in the vehicle. Lower concentrations were prepared by 2-fold dilutions. The pH of the treatment medium was measured and adjusted at concentrations ≥82.0 μg/mL using 1N hydrochloric acid (HCl; CAS No. 7647-01-0, Lot No. RNBD4214, Exp. Date: 31 Mar 2018). No pH adjustment was necessary to maintain neutral pH in the treatment medium at the remaining concentrations tested.
The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 115.63 and 92.21% at a concentration of 1312 μg/mL with and without S9, respectively. Based upon these results, the concentrations selected for the definitive mutagenicity assays were 82, 164, 328, 656 and 1312 μg/mL with and without S9.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive controls induced significant increases in mutant frequency (p < 0.01)
- Negative (solvent/vehicle) historical control data: No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p > 0.01).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: adjusted relative survival

Conclusions:

Under the conditions of the assay described in this report, the test substance was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A key, K1 micronucleus test with the test substance is available. Under the conditions of the assay, the test substance was concluded to be negative for the induction of micronucleated polychromatic erythrocytes.

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:

12 January 2018 to 09 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Updated guidance adopted 29 July 2016

Deviations:

no

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

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

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

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

Dose / conc.:

42.5 mg/kg bw/day (nominal)

Remarks:

Group 2
Sampling time: 24h after administration

Dose / conc.:

85 mg/kg bw/day (nominal)

Remarks:

Group 3
Sampling time: 24h after administration

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

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.

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.

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

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

Three reliable, key in vitro studies are available.

Ames:

The test article was tested in the bacterial reverse mutation assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA 1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. Sterile water for injection was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article formed a clear solution in sterile water for injection at approximately 50 mg/mL , the maximum concentration tested in the solubility test. In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Neither precipitate nor background lawn toxicity were observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 µg per plate. In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 µg per plate. Neither precipitate nor toxicity were observed.

The results of the bacterial reverse mutation assay indicate that, under the conditions of this study, the test item did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

HPRT

The test substance was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6 -thioguanine (TG resistance, TGr). Water was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 2.56, 5.13, 10.3, 20.5, 41.0, 82.0, 164, 328, 656 and 1312 µg/mL. The maximum concentration evaluated was based on solubility limitations of the test substance in the vehicle. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 115.63 and 92.21% at a concentration of 1312 µg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 82, 164, 328, 656 and 1312 µg/mL with and without S9.

In the first definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. The average absolute cloning efficiency of vehicle controls was <60% (at initial survival and selection) for treatment condition without S9 activation. Therefore, this treatment condition was repeated with the same test concentrations. The average adjusted relative survival was 103.7% at a concentration of 1312 µg/mL with S9. Cultures treated at all concentrations with S9 were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with S9 (p>0.01). The positive controls induced significant increases in mutant frequency (p<0.01). In the second definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Due to technician error, incorrect number of cells was subcultured and lesser number of cells was plated for mutant selection. Therefore, this trial (without S9) was repeated with the same test concentrations as the first mutagenicity assay. In the final definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. The average adjusted relative survival was 83.83% at a concentration of 1312 µg/mL without S9. Cultures treated at all concentrations without S9 were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p>0.01). The positive controls induced significant increases in mutant frequency (p<0.01).

Under the conditions of the assay, the test substance was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.

Chromosome aberration:

The test substance was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster ovary (CHO) cells in both the absence and presence of an exogenous metabolic activation system. CHO cells were treated for 4 hours in the absence and presence of S9, and for 20 hours in the absence of S9 (Roy, 2019). Water was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.131 to 1310 µg/mL (10 mM), which was the limit dose for this assay. Cytotoxicity (>=50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non‑activated and S9-activated 4-hour exposure groups. Cytotoxicity was observed at doses >=39.3 µg/mL in the non-activated 20-hour exposure group. Based upon these results, the doses chosen for the chromosome aberration assay ranged from 10 to 1310 µg/mL for the non‑activated and S9-activated 4-hour exposure groups and from 5 to 50 µg/mL for the non‑activated 20-hour exposure group.

In the chromosome aberration assay, required cytotoxicity (50 to 60% reduction in cell growth index relative to the vehicle control) was not observed up to 10 mM in the non-activated and S9-activated 4-hour exposure groups. In non-activated 20-hour exposure group greater than 50% cytotoxicity was observed ≥ 45 μg/mL. The doses selected for evaluation of chromosome aberrations were 325, 650, and 1310 μg/mL for the non-activated and S9-activated 4-hour exposure groups. The top concentration in either condition was the limit top dose (10 mM). In non-activated 20-hour exposure group concentrations of 5, 25, and 50 μg/mL was selected for evaluation of chromosome aberration. Top concentration in 20-hour exposure group was based on required cytotoxicity. As per testing guideline, the top concentration evaluated for chromosome aberration must be limit dose (10 mM) or required cytotoxicity (50 to 60% reduction in cell growth).

OECD testing guideline 473 (2016) primarily requires evaluation of structural chromosome aberrations, although polyploidy and endoreduplication are also evaluated that is an indicator of numerical aberrations. As per guideline, test substance is considered positive if one or more concentration show (i) statistically significant induction in chromosome aberration, (ii) induction is dose responsive (iii) induced values are outside the upper limit of 95% control limit of historical data. If these three conditions are not met, the results are negative.

In the non-activated and S9-activated 4-hour exposure groups, no significant or dose-dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed at any dose (Fisher’s Exact test = p > 0.05 and Cochran-Armitage test = p > 0.05).

In the non-activated 20-hour exposure group, statistically significant and dose-dependent increases in structural aberrations (5.0% and 7.3%) were observed at doses 25 and 50 μg/mL (Fisher’s Exact test = p ≤ 0.01 and Cochran-Armitage test = p ≤ 0.05). No significant or dose-dependent increases in numerical aberrations were observed in the non-activated 20 -hour exposure group (Fisher’s Exact and Cochran-Armitage tests = p > 0.05). Therefore, the statistically significant increase in structural aberrations was considered equivocal.

In order to confirm the positive response observed in the non-activated 20-hour exposure group, 4 additional dose levels of 30, 35, 40, and 45 μg/mL were included in the microscopic evaluation. Statistically significant and dose-dependent increases in structural aberrations (5.0%, 6.0%, 6.3%, and 4.7%) were observed at doses 30, 35, 40, and 45 μg/mL, respectively (Fisher’s Exact test = p ≤ 0.05 and Cochran-Armitage tests = p ≤ 0.01). No significant or dose-dependent increases in numerical aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicate 3-Butoxypropylamine was positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in the absence of the exogenous metabolic activation system. 3-Butoxypropylamine was negative for the induction of structural and numerical chromosome aberrations in the presence of the exogenous metabolic activation system. Hence, based on the results of this study 3-Butoxypropylamine is considered to be clastogenic in the non-activated test system in the in vitro mammalian chromosome aberration test using CHO cells.

Genetic toxicity in vivo:

A key, K1 in vivo micronucleus test performed with the test substance according to the test guideline OECD 474 is available (Young, 2019). The test substance was evaluated for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocytes (PCEs) in rat bone marrow. Deionized water was used as the vehicle. Test and/or control substance formulations were administered once at a dose volume of 10 mL/kg by oral gavage. In the dose range-finding assay (DRF), male and female rats were dosed at 170 and 85 mg/kg. Based on results of the DRF, the high dose for the definitive assay was 170 mg/kg, which was estimated to be the maximum tolerated dose (MTD). The definitive assay dose levels tested were 42.5, 85 and 170 mg/kg in male rats. Twenty four hours after the last dose, animals were euthanized and bone marrow collected and processed for the micronucleus assay. Additional animals were dosed in the vehicle and high dose group of 170 mg/kg and these were harvested 48 hours after dosing. No statistically significant increase in the incidence of MnPCEs in the test substance treated groups was observed relative to the vehicle control groups. The positive control induced a statistically significant increase in the incidence of MnPCEs. The number of MnPCEs in the vehicle control groups did not exceed the historical control range.

Under the conditions of this study, the oral administration of the test substance at doses up to and including a dose of 170 mg/kg was concluded to be negative in the Micronucleus assay.

Justification for classification or non-classification

Based on the results and according to the CLP regulation, the test substance is not to be classified as mutagenic.