4-[3-(1-naphthylamino)propyl]morpholine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity and Mammalian clastogenicity assays in vitro

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:

2016

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

a metabolically active extract of rat liver (treated with Aroclor 1254)

Test concentrations with justification for top dose:

31.25, 62.5, 125, 250, 500 and 1000 µg 4-(3-(1-Naphthylamino)propyl)morpholine/plate both in the absence and presence (10% v/v S9 mix) of metabolic activation
Basis for top dose selection was inhibition of the bacterial lawn at 1500 and 5000 µg in the initial toxicity mutation assay.

Vehicle / solvent:

Dimethyl sulphoxide

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene (613-13-8)

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Negative Controls

The results of the study indicate that the revertant colony numbers for the negative controls (DMSO) in all strains were within limits of historical range.

Positive Controls

2-Aminoanthracene was used as the positive control in the presence of metabolic activation for all the tester strains during the Initial Toxicity Mutation Assay and Confirmatory Mutation Assay. Historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of metabolic activation.

Positive controls (both in the absence and presence of metabolic activation during both the trials) exhibited a clear increase in the number of revertants when compared with the concurrent negative control and were within the historical ranges .This demonstrated the efficiency of the test system and suitability of the procedures employed in the assay.

An increase in the mean number of revertants was not observed in Salmonella typhimurium tester strain TA100 (Initial Toxicity Mutation Assay and Confirmatory Mutation Assay) treated with 2-aminoanthracene in the absence of metabolic activation, but a clear increase was observed in the presence of metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.

Initial Toxicity Mutation Assay

Bacterial cultures were exposed to 4-(3-(1-Naphthylamino)propyl)morpholine at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (two plates/concentration) both in the absence and presence of metabolic activation (5% v/v S9 mix). Precipitation was not observed up to the tested concentration of 5000 µg/plate. Complete inhibition of background lawn was observed at tested concentrations 1500 and 5000 µg/plate in all the tester strains both in absence and presence of metabolic activation. Partial inhibition of background lawn was observed at tested concentration of 500 µg/plate in all the tester strains both in absence and presence of metabolic activation. No inhibition of background lawn was observed up to the tested concentration of 150 µg/plate in all the tester strains both in absence and presence of metabolic activation. Reduction in number of revertant colonies of 44-56 % in the absence of metabolic activation in all the tester strains and 48-54 % [TA1535, TA98, TA100, and Escherichia coli WP2 uvrA (pKM101)] in the presence of metabolic activation (5% v/v S9 mix) was observed at tested concentration of 500 µg/plate. In tester strain TA1537, no appreciable reduction in revertant colonies was observed at the tested concentration of 500 µg/plate in the presence of metabolic activation (5% v/v S9 mix). Normal growth was observed up to the tested concentration of 150 µg/plate in all tester strains both in the absence and presence of metabolic activation (5% v/v S9 mix). No increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control. Hence, 1000 µg 4-(3-(1-Naphthylamino)propyl)morpholine/plate was selected as the highest concentration to be tested in the Confirmatory Mutation Assay both in the absence and presence of metabolic activation (10% v/v S9 mix).

Confirmatory Mutation Assay

In the Confirmatory Mutation Assay, bacterial cultures were exposed to 4-(3-(1-Naphthylamino)propyl)morpholine at concentrations of 31.25, 62.5, 125, 250, 500 and 1000 µg/plate (three plates/concentration) both in the absence and presence of metabolic activation (10% v/v S9 mix). Precipitation was not observed up to the tested concentration of 1000 µg/plate. Partial inhibition of background lawn was observed at tested concentrations of 500 and 1000 µg/plate in all the tester strains both in absence and presence of metabolic activation. No inhibition of background lawn was observed up to the tested concentration of 250 µg/plate in all the tester strains both in absence and presence of metabolic activation. Reduction in number of revertant colonies of 63-72 % in the absence of metabolic activation and 52-70 % in the presence of metabolic activation (10% v/v S9 mix) was observed at tested concentration of 1000 µg/plate in all the tester strains. Reduction in number of revertant colonies of 43-50 % in the absence of metabolic activation and 47-49 % in the presence of metabolic activation (10% v/v S9 mix) was observed at tested concentration of 500 µg/plate in all the tester strains.

Normal growth was observed up to the tested concentration of 250 µg/plate in all tester strains. No increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

Conclusions:

From the results of this study, under the specified experimental conditions, 4-(3-(1-Naphthylamino)propyl)morpholine was concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2 uvrA (pKM101).

Executive summary:

The potential of 4-(3-(1-Naphthylamino)propyl)morpholine to induce reverse mutations in Salmonella typhimurium strains TA1537, TA1535, TA98 and TA100 anda tryptophan deficient strain, Escherichia coli WP2uvrA (pKM101) was evaluated in the bacterial reverse mutation test using the pre-incubation method.

 

4-(3-(1-Naphthylamino)propyl)morpholinewas tested in the absence and presence of metabolic activation using dimethyl sulfoxide (DMSO) as the solvent. In the Initial Toxicity Mutation Assay, bacterial cultures were exposed to 4-(3-(1-Naphthylamino)propyl)morpholine at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (two plates/concentration). Precipitation was not observed up to the concentration of 5000 µg/plate. Complete inhibition of background lawn was observed at the tested concentrations of 1500 and 5000 µg/plate in all the tester strains both in absence and presence of metabolic activation (5% v/vS9 mix). Partial inhibition of background lawn was observed at tested concentration of 500 µg/plate in all the tester strains both in absence and presence of metabolic activation. Normal background lawn pattern without reduction in number of revertant colonies was observed up to the tested concentration of 150 µg/plate in all the tester strains in both absence and presence of metabolic activation (5% v/vS9 mix). No increase in the number of revertant colonies was observed in any of the tester strains with the test item at any of the tested concentrationsin the absence or presence ofmetabolic activationwhen compared with the concurrent negative control. 

 

In the Confirmatory Mutation Assay, bacterial cultures were exposed to 4-(3-(1-Naphthylamino)propyl)morpholine at concentrations of 31.25, 62.5, 125, 250, 500 and 1000 µg/plate (three plates/concentration) both in the absence and presence of metabolic activation (10% v/vS9 mix). Precipitation was not observed up to the concentration of 1000 µg/plate. Partial inhibition of background lawn was observed at the tested concentrations of 500 and 1000 µg/plate in all the tester strains both in absence and presence of metabolic activation. No inhibition of background lawn was observed up to the tested concentration of 250 µg/plate in all the tester strains in both absence and presence of metabolic activation. No increase in the number of revertant colonies was observed in any of the tester strains with the test item at any of the tested concentrations in the absence or presence of metabolic activation when compared with the concurrent negative control.

 

All the values for the negative controls were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

 

The 4-(3-(1-Naphthylamino)propyl)morpholine stock concentrations 312.5, 625, 1250, 2500, 5000 and    10000 µg/mL were found to be within acceptable range of ± 15% of nominal concentrations during the Confirmatory Mutation Assay. The 0 hour concentrations of 4-(3-(1-Naphthylamino)propyl)morpholine in the dose formulation were found to be 108%, 106%, 107%, 103%, 97.4%, 96.9% of the nominal concentrations of dose level T1 (312.5 µg/mL), T2 (625 µg/mL), T3 (1250 µg/mL), T4 (2500 µg/mL), T5 (5000 µg/mL) and T6 (10000 µg/mL), respectively. The concentrations of 4-(3-(1-Naphthylamino)propyl)morpholine in the dose formulation after 4 hours were found to be 99.3% and 99.1% of the 0 hour concentrations of dose level T1 (312.5 µg/mL) and T6 (10000 µg/mL), respectively. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient.

 

All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, 4-(3-(1-Naphthylamino)propyl)morpholine is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2uvrA (pKM101).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Chromosomal aberration assay utilizing rat lymphocytes.

Specific details on test material used for the study:

Test Material Name: 4-(3-(1-naphthylamino)propyl)morpholine
Chemical Name: N-1-Naphthalenyl-4-morpholinepropanamine
Synonyms: None
Lot/Reference/Batch Number: ZA01212016
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 94.4% by liquid chromatography with identification by nuclear magnetic resonance spectroscopy and liquid chromatography mass spectrometry (Ferrer, 2016a).
Test Material Stability Under Storage Conditions: 4-(3-(1-Naphthylamino)propyl)morpholine, lot ZA01212016, was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under USEPA OPPTS Guideline 830.6313 (Ferrer, 2016b).

Target gene:

Chromosomal aberration assay utilizing rat lymphocytes.

Species / strain / cell type:

lymphocytes: Rat

Details on mammalian cell type (if applicable):

Species and Sex: Rats (Male)
Strain and Justification: Crl:CD(SD) Rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.
Supplier and Location: Charles River (Kingston, New York)
Age at Study Start: 10-16 weeks

Lymphocyte Cultures:
The animals were euthanized with carbon dioxide just prior to collecting the samples by cardiac puncture. Blood, treated with an anticoagulant (e.g., heparin) from several rats, was pooled for sample collection. Whole blood cultures were set up in complete medium (RPMI 1640 medium with 25 mM HEPES, supplemented with 10% heat-inactivated fetal bovine serum, antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone 0.25 μg/ml), and an additional 2 mM L-glutamine) in addition with 30 μg/ml PHA-P. Cultures were initiated by inoculating approximately 0.5 ml of whole blood into 5 ml of medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C. Treatment medium was the above-mentioned medium without serum and was used during the 4-hour treatment conditions, while complete medium was used for the 24-hour treatment condition.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate prepared from Aroclor 1254-treated (500 mg/kg body weight) male Sprague Dawley rats.

Test concentrations with justification for top dose:

Cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 677.8 μg 4-(3-(1-naphthylamino)propyl)morpholine per ml of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO, CAS No. 67-68-5) was selected as the solvent used to dissolve the test material and was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Cyclophosphamide monohydrate

Details on test system and experimental conditions:

Lymphocyte Cultures:
The animals were euthanized with carbon dioxide just prior to collecting the samples by cardiac puncture. Blood, treated with an anticoagulant (e.g., heparin) from several rats, was pooled for sample collection. Whole blood cultures were set up in complete medium (RPMI 1640 medium with 25 mM HEPES, supplemented with 10% heat-inactivated fetal bovine serum, antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone 0.25 μg/ml), and an additional 2 mM L-glutamine) in addition with 30 μg/ml PHA-P. Cultures were initiated by inoculating approximately 0.5 ml of whole blood into 5 ml of medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C. Treatment medium was the above-mentioned medium without serum and was used during the 4-hour treatment conditions, while complete medium was used for the 24-hour treatment condition.

In Vitro Metabolic Activation System:
S9 liver homogenate prepared from Aroclor 1254-treated (500 mg/kg body weight) male Sprague Dawley rats were purchased from a commercial source and stored at -100°C or below. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a "mix" (O'Neill et al., 1982). The mix consisted of 10 mM MgCl2·6H2O, 5 mM glucose-6-phosphate, 4 mM nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 30 mM KCl, and 50 mM sodium phosphate (pH 8.0). The reconstituted mix was added to the treatment medium to obtain the desired final concentration of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the co-factors in the medium was 1/5 of the concentrations stated above.

Preparation of the Treatment Solution and Administration of the Test Material:
The test material was found to be soluble in DMSO up to 271.1 mg/ml. All test material solutions were prepared fresh on the day of treatment and used within one hour of preparation. The test material was dissolved in DMSO and further diluted (1: 100) in medium. This technique has been shown to be an effective method for detecting various in vitro clastogens in this test system. All dosing units were expressed in μg/ml. MMC was dissolved in treatment medium and CP stock was dissolved in distilled water.

Dose Level Selection:
The cultures were treated with various concentrations of the test material and the selected concentration of the positive control chemicals. Soluble materials were tested up to 10 mM, 2000 μg/ml, or 2 μl/ml, whichever was the lowest. Test materials with limited solubility were tested up to or beyond their limit of solubility. In some cases, more than one insoluble concentration was tested to ascertain whether toxicity would occur at higher insoluble concentrations. The other concentrations tested were separated by a factor of 2 to 3.

Analytical Verification of Dosing Solutions:
The selected concentrations of the test material in the stock dosing solutions used for treatment in Assay A1 were verified by the Analytical Chemistry Laboratory, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples were diluted in an appropriate solvent and analyzed by high performance liquid chromatography with ultraviolet detection (HPLC/UV). Analytical method validation was performed concurrently with sample analysis and was described in the final report. Homogeneity analysis was not conducted as dosing solutions were not administered as suspensions.

Identification of the Test System:
All test cultures were identified using self-adhesive labels containing a code system that identified the test material, experiment number, treatment, and replicate.

Evaluation criteria:

Evaluation Criteria:
For a test to be acceptable, the chromosomal aberration frequency in the positive control cultures should be significantly higher than the vehicle controls. The aberration frequency in the vehicle and positive controls should be within the control limits of the laboratory historical control values as calculated using previous laboratory values. A test chemical was considered positive in this assay if it induced a statistically significant, dose-related increase in the frequency of cells with aberrations and the incidence of aberrant cells was outside the control limits of the laboratory historical vehicle control range. A test chemical was considered negative in this assay if it did not induce a statistically significant, dose-related increase in the frequency of cells with aberrations and the incidence of aberrant cells was not outside the control limits of the laboratory historical vehicle control range. If a test chemical did not meet either of the above criteria it may have been considered equivocal.

Statistics:

Statistical Analysis:
The proportions of cells with aberrations (excluding gaps) were compared by the following statistical methods. At each dose level, data from the replicates were pooled. A two-way contingency table was constructed to analyze the frequencies of aberrant cells. An overall Chi-square statistic, based on the table, was partitioned into components of interest. Specifically, statistics were generated to test the global hypothesis of no difference in the average number of cells with aberrations among the dose groups (Armitage, 1971). An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If this statistic was found to be significant at alpha = 0.05, pairwise tests (i.e., control vs. treatment) were performed at each dose level and evaluated at alpha = 0.05, versus a one-sided alternative. If any of the pairwise tests were significant, a test for linear trend of increasing number of cells with aberrations with increasing dose was performed (Armitage, 1971).
Polyploid cells were analyzed by the Fisher Exact probability test (Siegel, 1956). The number of polyploid cells were pooled across replicates for the analysis and evaluated at alpha = 0.05. The data was analyzed separately based on the presence or absence of S9 and based on the exposure time.

Species / strain:

lymphocytes: Rat

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No significant increases in the incidence of polyploid cells or frequency of cells with aberrations in the test material treated cultures (10.6, 42.4 and 169.5 μg/ml) as compared to the vehicle control.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: Rat

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No significant increases in the incidence of polyploid cells or frequency of cells with aberrations in the test material treated cultures (5.3, 21.2 and 42.4 μg/ml) as compared to the vehicle control.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: Rat

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Statistical analyses of these data did identify significant differences between the vehicle control and all of the treatment concentrations (i.e., 2.6, 5.3 and 10.6 μg/ml) with a positive linear trend (alpha = 0.05).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

pH and Osmolality:
The pH and osmolality of treatment medium containing approximately 2711.0 μg/ml of the test material and medium containing 1% DMSO were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts), respectively. There was no appreciable change in either the pH or osmolality at this concentration as compared to the treatment medium with solvent alone (treatment medium with the test material, pH = 7.36, osmolality = 404 mOsm/kg H2O; treatment medium with 1% DMSO, pH = 7.34, osmolality = 436 mOsm/kg H2O).

Remarks on result:

other: Assay A1 4-Hour Treatment

Assay A1:

Cultures were treated with the test material for 4 hours in the absence and presence of S9 activation at concentrations of 0 (vehicle control), 10.6, 21.2, 42.4, 84.7, 169.5, 338.9, and 677.8 μg/ml. Cultures were also treated continuously for 24 hours in the absence of S9 with the above concentrations plus an additional lower concentration of 5.3 μg/ml. The test material precipitated in the treatment medium at the top concentration (i.e., 677.8 μg/ml) in all treatment conditions, as observed at the end of treatment. Analytically detected concentrations of the test material in the stock solutions (Assay A1) varied from 93.0 to 100.7% of the target and verified that concentrations used for treatment were within the acceptable range.

Short Treatment:

In the absence of S9, the cultures displayed excessive toxicity at the two highest target concentrations (i.e., 338.9 and 677.8 μg/ml) as indicated by the lack of observable mitotic figures. The remaining cultures exhibited relative mitotic indices ranging from 43.2 to 91.0% compared to the vehicle control values. In the presence of S9, the cultures displayed excessive toxicity at the two highest target concentrations (i.e., 338.9 and 677.8 μg/ml) as indicated by the lack of observable mitotic figures. The remaining cultures exhibited relative mitotic indices ranging 18.4 to 49.0% as compared to the vehicle control values. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 10.6, 42.4, and 169.5 μg/ml were chosen for the determination of chromosomal aberration frequency and incidence of polyploidy in the absence of S9 activation. Due to the lack of a target concentration comparable to the vehicle control and no observable dose dependent cytotoxicity response, in the presence of S9, this portion of the assay was repeated in a separate assay.

Among the cultures treated with the positive control chemicals for 4 hours, 0.5 μg/ml of MMC was selected for evaluation of aberrations in the absence of S9.

There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values.

In the 4-hour non-activation assay, the frequency of cells with aberrations in the vehicle control was 0.3% and the corresponding values at treatment levels of 10.6, 42.4, and 169.5 μg/ml were 0.3, 0.7, and 1.0%, respectively. Statistical analyses of these data did not identify significant differences between the vehicle control and any of the treated cultures without or with S9 activation. The frequencies of aberrant cells observed in the test material treated cultures were within the control limits of the laboratory historical vehicle control range.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in MMC (-S9, 4-hour treatment cultures was 29.3%. All values were within the control limits of the laboratory historical positive control range.

Continuous Treatment:

Based upon the negative findings in the 4-hour treatment, slides from the continuous 24-hour treatment were evaluated. Cultures treated continuously for 24 hours in the absence of S9 activation exhibited excessive toxicity at the three highest target concentrations (i.e., 169.5, 338.9 and 677.8 μg/ml) as indicated by the lack of observable mitotic figures. The remaining cultures exhibited relative mitotic indices ranging from 23.7 to 94.9% relative to the vehicle control value. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 5.3, 21.2, and 42.4 μg/ml were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy. Cultures treated

with 0.075 μg/ml MMC were selected to serve as the positive control.

There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values.

The frequency of aberrant cells in the vehicle control was 0.7% and the corresponding values at concentration levels of 5.3, 21.2, and 42.4 μg/ml were 0.0, 0.3, and 0.0%, respectively. There were no statistically significant differences between the test material treated cultures and the vehicle control values and all values were within the control limits of the laboratory historical vehicle control range.

A significant increase in the frequency of cells with aberrations was observed in cultures treated with the positive control chemical. Aberrant cell frequency in MMC treated cultures was 18.0%. All values were within the control limits of the laboratory historical positive control range.

Assay B1:

Cultures were treated with the test material for 4 hours in the presence of S9 activation at concentrations of 0 (vehicle control), 2.6, 5.3, 10.6, 21.2, 42.4, 84.8, and 169.5 μg/ml.

Short Treatment:

In the presence of S9, the cultures displayed excessive toxicity at the two highest target concentrations (i.e., 84.8 and 169.5 μg/ml) as indicated by the lack of observable mitotic figures. The remaining cultures exhibited relative mitotic indices ranging from 21.0 to 69.4% compared to the vehicle control values. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 2.6, 5.3, and 10.6 μg/ml were chosen for the determination of chromosomal aberration frequency and incidence of polyploidy in the presence of S9 activation. Although the relative mitotic index of the lowest concentration (i.e., 2.6 μg/ml) was slightly lower than the corresponding control value, the variability in

response between assays (A1 and B1) and the concentration response in assay B1 indicated an acceptable assay performance.

Among the cultures treated with the positive control chemicals for 4 hours, 4.0 μg/ml of CP was selected for evaluation of aberrations in the presence of S9.

There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values.

In the 4-hour activation assay (Table 9), the frequency of cells with aberrations in the vehicle control was 0.0% and the corresponding values at treatment levels of 2.6, 5.3, and 10.6 μg/ml were 2.7, 3.7, and 4.4%, respectively. As a result of the decreased relative mitotic index and low mitotic figures present on the slides, only 271 metaphase spreads were evaluated from the 10.6 μg/ml concentration, instead of the standard 300. This minor variation did not have any impact on the assay results interpretation. Statistical analyses of these data did identify significant differences between the vehicle control and all of the treatment concentrations (i.e., 2.6, 5.3, and 10.6 μg/ml) with a positive linear trend (alpha = 0.05). The

frequencies of aberrant cells observed in the test material treated cultures were outside the control limits of the laboratory historical vehicle control range.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in CP (+S9, 4-hour treatment cultures was 20.9%. All values were within the control limits of the laboratory historical positive control range.

Conclusions:

It was concluded that under the experimental conditions used, 4-(3-(1-naphthylamino)propyl)morpholine was considered clastogenic in this in vitro chromosomal aberration assay utilizing rat lymphocytes as a result of the clear positive
response observed in the 4 hour treatment (with S9).

Executive summary:

4-(3-(1-naphthylamino)propyl)morpholine (N-1-Naphthalenyl-4-morpholinepropanamine) was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes.

Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 677.8 μg 4-(3-(1-naphthylamino)propyl)morpholine per ml of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium. The analytically determined concentrations of 4-(3-(1-naphthylamino)propyl)morpholine in the dose preparations ranged from 93.0 to 100.7% of the targeted values. The duration of treatment was 4 hours without and with S9 and 24 hours without S9. Selection of concentrations for the determination of the incidence of chromosomal aberrations was based upon cytotoxicity of the test material. In this study cultures treated for 4 hours with targeted concentrations of 0 (vehicle control), 10.6, 42.4, and 169.5 μg/ml in the absence of S9, 0 (vehicle control), 2.6, 5.3, and 10.6 μg/ml in the presence of S9, and cultures treated for 24 hours with targeted concentrations of 0 (vehicle control), 5.3, 21.2, and 42.4 μg/ml were analyzed for chromosomal aberration frequency.

There were no significant increases in the frequency of cells with aberrations administered 4-(3-(1-naphthylamino)propyl)morpholine in the absence of S9 activation. There was a significant increase in the frequency of cells with aberrations in the presence of S9, with 0.0% in the vehicle control and 2.7, 3.7, and 4.4% in the treatment concentrations (i.e., 2.6, 5.3, and 10.6 μg/ml), respectively. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of aberrant cells. The vehicle and positive controls in all assays were within the acceptable historical ranges and fulfilled the requirements for a valid assay.

Based upon these results, 4-(3-(1-naphthylamino)propyl)morpholine was considered to be positive for clastogenicity in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

OECD 474 n vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus assay

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:

2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Identification: 4-[3-(1-naphthylamino)propyl]morpholine
Alternate Identification: N-1-napthalenyl-4-morpholinepropanamine
Molecular Weight: 270.4
CAS No.: 5235-82-5
Lot No.: ZA01212016
Recertification Date: 14 Mar 2018; 24 Jun 2023
Physical Description: Brown powder
Purity: 94.4%; 96.58%
Correction Factor: 1.0582
Storage Conditions: 18°C to 24°C
Provided by: Sponsor

A preliminary characterization of purity of the test substance was assessed by the Sponsor in 2021 and was assigned with a purity of 94.5%. The same lot of the test substance was also previously characterized in 2016 and was observed with a purity of 94.4% (Ferrer, 2016, Study No. ML AL-2016-002085). The same lot of the bulk test substance was evaluated for purity after completion of this study in Jun 2022, and the purity was determined to be 96.58%. The formulations prepared during the study were corrected for purity using a correction factor of
1.0582 based on initial characterization (94.5%) provided by the Sponsor. The slight increase in purity did not impact the nominal dose levels used on the study, as the difference in the purity determined before the study and after the study was minimal.

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

At this time, studies in laboratory animals provide the best available basis for extrapolation to
humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
The Sprague Dawley rat was chosen as the animal model for this study as it is an accepted rodent
species for nonclinical toxicity testing by regulatory agencies.
The total number of animals used in this study was considered to be the minimum required to
properly characterize the effects of the test substance. This study was designed such that it did
not require an unnecessary number of animals to accomplish its objectives.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Receipt:
On 19 Jan 2022, 25 Jan 2022, and 01 Feb 2022, Crl:CD(SD) rats were received from Charles
River Laboratories, Inc., Raleigh, NC. The animals were approximately 7–11 weeks old and
weighed between 157 and 424 g (males) (see Appendix 1), and between 164 and 250 g (females)
at the initiation of dosing.

Environmental Acclimation:
Each animal was inspected by qualified personnel upon receipt. Animals judged to be in good
health were placed in acclimation for at least 5 days.

Selection, Assignment, and Disposition of Animals:
Animals were assigned to groups by a stratified randomization scheme designed to achieve
similar group mean body weights. Males and females were randomized separately. Animals at
extremes of body weight range were not assigned to groups. Individual body weights at
randomization were within ± 20% of the mean for each sex.
The disposition of all animals was documented in the study records.

Housing:
Housing: Single
Caging: Solid-bottom cages containing appropriate bedding material (Bed-O-Cobs®or other suitable material).
Cage Identification: Color-coded cage card indicating study, group, animal number(s), and
sex.
Housing set-up was as specified in the Guide for the Care and Use of Laboratory Animals
(National Research Council, 2011). Animals were separated during designated
procedures/activities. Cages were arranged on the racks in group order. Where possible, control
group animals were housed on a separate rack from the test substance-treated animals.

Environmental Conditions:
The targeted conditions for the animal room environment were as follows:
Temperature: 68°F to 78°F (20°C to 26°C)
Humidity: 30% to 70%
Light Cycle: 12 hours light and 12 hours dark (except during designated procedures).

Food:
Diet: PMI Nutrition International, LLC Certified Rodent LabDiet 5002.
Type: Meal
Frequency: Ad libitum.
Analysis: Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Testing Facility. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water:
Type: Municipal tap water, treated by reverse osmosis and ultraviolet irradiation.
Frequency/Ration: Freely available to each animal via an automatic watering system. Water
bottles were provided, if required.
Analysis: Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

Route of administration:

oral: gavage

Vehicle:

Identification: Propylene glycol, USP/FCC
CAS No.: 57-55-6
Lot No.: 206214
Retest Date: Nov 2023
Storage Conditions: 18°C to 24°C
Provided by: Testing Facility

Details on exposure:

Administration of Test Materials:
Test substance formulations and vehicle control were administered once daily, for 2 consecutive days via oral gavage. The dose formulations were stirred continuously at room temperature
during dosing and as possible during transport. Each dose was administered via a syringe
equipped with attached gavage cannula.
Doses were based on the most recent body weight measurement. The first day of dosing was
based on Day 1 body weights.

Duration of treatment / exposure:

Test substance formulations and vehicle control were administered once daily, for 2 consecutive days via oral gavage.

Frequency of treatment:

Test substance formulations and vehicle control were administered once daily, for 2 consecutive days via oral gavage.

Dose / conc.:

20 mg/kg bw/day

Remarks:

Positive control: Positive control substance reference slides, obtained from a separate experiment performed at the Test Site with cyclophosphamide monohydrate administered once approximately 24 hours prior to harvest, were included in the slide set to verify scorer proficiency (Roy, 2021, 30810).

Dose / conc.:

1 200 mg/kg bw/day

Remarks:

Micronucleus Assay: 6 Females
Toxicokinetic Phase: 3 Females

Dose / conc.:

600 mg/kg bw/day

Remarks:

Micronucleus Assay: 6 Females

Dose / conc.:

300 mg/kg bw/day

Remarks:

Micronucleus Assay: 6 Females

Dose / conc.:

800 mg/kg bw/day

Remarks:

Micronucleus Assay: 6 Males
Toxicokinetic Phase 3 Males

Dose / conc.:

400 mg/kg bw/day

Remarks:

Micronucleus Assay: 6 Males

Dose / conc.:

200 mg/kg bw/day

Remarks:

Micronucleus Assay: 6 Males

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle: Micronucleus Assay - 6 Males and 6 Females

No. of animals per sex per dose:

See "Study Design" document attached for details on dosing regimen.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive Control Article: Cyclophosphamide monohydrate in Ultrapure water
Positive control substance reference slides, obtained from a separate experiment performed at the Test Site with cyclophosphamide monohydrate administered once approximately 24 hours prior to harvest, were included in the slide set to verify scorer proficiency (Roy, 2021, 30810).

Tissues and cell types examined:

Micronucleus Evaluation:
Bone Marrow Sample Collection:
Bone marrow was collected from up to 5 surviving animals in each group at the time of euthanasia for the terminal necropsy from the right femur of animals euthanized by inhalation of carbon dioxide. Five animals/group in the vehicle control and test substance-treated groups were euthanized 18 to 24 hours following the last dose administration. The sixth animal was euthanized via carbon dioxide inhalation and the carcasses were discarded. Bone marrow was aspirated or flushed 2 to 3 times from the right femur into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS).

Details of tissue and slide preparation:

Micronucleus Evaluation:
Bone Marrow Sample Processing:
The bone marrow was centrifuged and all but approximately 0.25 mL (or a volume approximately twice that of the cell pellet) of HI FBS was decanted, and the pellet was resuspended in the remaining HI FBS. Bone marrow smears were prepared by placing approximately 1 drop of cell suspension onto a minimum of 4 appropriately labeled, clean microscope slides. Each slide was coded so that the treatment group would not be revealed during subsequent analysis. The slides were air dried, fixed in 100% methanol for approximately 20 minutes and allowed to air dry a second time. The slides were stored and shipped at ambient temperature to Charles River Skokie and then to the Test Site due to a change in the protocol.

Bone Marrow Sample Analysis:
Prior to analysis, the coded slides were stained with acridine orange staining solution (Hayashi et al., 1983). Two separate evaluations were made for each slide: 1) 500 total erythrocytes (TE:PCEs + normochromatic erythrocytes [NCs]) were counted per animal and the PCE:TE ratio was determined; and 2) the number of micronucleated PCEs (MN-PCEs) in total of 4000 PCEs scored per animal was determined.

Statistics:

STATISTICAL ANALYSIS:
Statistical analyses were not performed due to the absence of a concurrent control group for the
range-finding phase. However, means and standard deviations were calculated.

Key result

Sex:

female

Genotoxicity:

negative

Remarks:

All dose levels tested.

Toxicity:

yes

Remarks:

Test substance-related lower body weights were observed at 1200 mg/kg/day in females. Decreases in body temperatures were observed at ≥ 800 mg/kg/day in females.

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Based on effects on survival in both phases, the maximum tolerated dose (MTD) level was considered to be 800 mg/kg/day for females.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

All dose levels tested.

Toxicity:

yes

Remarks:

Test substance-related lower body weights were observed at ≥ 400 mg/kg/day in males. Decreases in body temperatures were observed at ≥ 800 mg/kg/day in males.

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Based on effects on survival in both phases, the maximum tolerated dose (MTD) level was considered to be 800 mg/kg/day for males.

Additional information on results:

Range-Finding Phase:
Mortality:
Test substance-related effects on survival were observed at ≥ 1200 mg/kg/day in males and at
2000 mg/kg/day in females, during the range-finding phase. Two males and 1 female at
2000 mg/kg/day and 1 male at 1200 mg/kg/day were found dead prior the scheduled euthanasia.
In the 2000 mg/kg/day group, Male Nos. 10002 and 10003 were found dead on Day 1 after dose
administration and were both noted with low carriage, slight incoordination, hunched posture,
and decreased activity at 1–3 hours post-dose. Female No. 10502 was found dead prior to the
scheduled euthanasia on Day 3, and was noted with clinical observations of decreased activity,
low carriage and slight incoordination, partly closed eyes, abnormal soft feces, erected fur,
urogenital brown fur staining attributed to the color of the test substance, hunched posture
between Days 1 and 2 on the days of dose administration.
In the 1200 mg/kg/day group, Male No. 9001 was found dead on Day 1 after dose administration
and was noted with observations of slight incoordination, low carriage, and decreased activity
after dose administration. Additionally, the animal was observed to be weak and lying on side
before being found dead.
All other animals survived to the scheduled euthanasia.

Clinical Observations:
Test substance-related clinical observations were noted at ≥ 200 mg/kg/day in females and at
≥ 400 mg/kg/day in males.
At 2000 mg/kg/day, test substance-related observations included decreased activity, hunched
posture, low carriage, abnormal slightly soft feces, and brown urogenital fur staining attributed to
the color of the test substance in both males and females. In addition, slight incoordination, and
erected fur in the females was observed.
At 1200 mg/kg/day, test substance-related observations of decreased activity, low carriage, and
hunched posture were noted in both males and females. In addition, males were observed with
slight incoordination, and females were observed with skin pallor, brown urogenital fur staining
attributed to the color of the test substance and abnormal slightly soft feces.
At 800 mg/kg/day, test substance-related ploughing behavior was observed in both males and
females at the time of dose administration. In addition, females were observed with brown
staining and wet fur in the urogenital region consistent with the color of the test substance, and
abnormal soft feces during the study.
At 400 mg/kg/day, test substance-related ploughing behavior observed in both males and females at the time of dosing. Additionally, abnormal slightly soft feces in a single male was also noted.
At 200 mg/kg/day, test substance-related ploughing behavior was noted on Day 2 at the time of
dosing in a single female.

Body Weights and Body Weight Gains:
Test substance-related body weight losses, compared to pretreatment body weights on Days 1–3,
were observed at dose levels ≥ 400 mg/kg/day in males and ≥ 200 mg/kg/day in females.
In the 400, 800, 1200, and 2000 mg/kg/day group males, mean body weight losses of -11.3 g,
-9.3 g, -42.5 g, and -25.0 g respectively were observed between Days 1–3. On Day 3, the mean
body weights in the 400, 800, 1200, and 2000 mg/kg/day group males were 6.5%, 5.0%, 11.9%,
and 6.5% decreased, respectively, compared to mean body weights on Day 1 and were associated
with the observed body weight losses. There were no test substance-related effects on body
weight in the 200 mg/kg/day group males.
In the 200, 400, 800, 1200, and 2000 mg/kg/day group females, mean body weight losses of
-10.7 g, -4.0 g, -14.0 g, -23.7 g, and -27.5 g, respectively were observed between Days 1–3. On
Day 3, the mean body weights in the 200, 400, 800, 1200, and 2000 mg/kg/day group females
were 6.1%, 2.3%, 8.2%, 10.1%, and 6.8% decreased, respectively, compared to mean body weights on Day 1 and corresponded to the body weight losses.

Body Temperature:
Test substance-related effects on body temperature were noted at ≥ 200 mg/kg/day in both males and females.
Mean body temperatures prior to dose administration on Days 1 and 2 in males and females
ranged between 34°C and 39°C. On Day 1, a ≥ 1°C decrease in mean body temperature was
observed at 2 and 5 hours post-dose in all test substance-administered males and female groups. A dose response was evident with a higher magnitude of approximately 2–4°C decreases in mean
body temperature observed at ≥ 800 mg/kg/day in males and females. On Day 2, mean body
temperatures at 2 and 5 hours post-dose were similar or noted to have a ≤ 1°C decrease after test
substance administration in the surviving males. However, in females, a higher magnitude of test
substance-related effects with an approximately 1–2°C decrease in mean body temperatures at 2
and/or 5 hours post-dose on Day 2 were observed in the 800, 1200, and 2000 mg/kg/day group
females.
Overall, a qualitative comparison of body temperatures after test substance administration to
pre-dose body temperatures showed a dose-dependent trend with higher magnitude of decreased
body temperatures observed at 1200 and 2000 mg/kg/day in males and at 800, 1200, and
2000 mg/kg/day in females.

Definitive Phase:
Mortality:
Test substance-related effects on survival were observed at 1200 mg/kg/day.
Female Nos. 13504 and 13502 in the 1200 mg/kg/day group were found dead on Day 2 and 3,
respectively. Both females were noted with decreased activity and low carriage at 1–3 hours
postdose on Day 1. In addition, Female No. 13502, was observed soft feces (moderate) with
brown anal fur staining on Day 2, and Female No. 13504 was observed with moderate
incoordination and abnormal gait on Day 1. There were no macroscopic observations at necropsy
in both animals.
All other animals survived to the scheduled euthanasia.

Clinical Observations:
Test substance-related clinical observations were noted in all test substance-administered males
and females.
Test substance-related observations included hunched posture in all test substance-administered
female groups, low carriage, moderate incoordination and decreased activity in 600 and
1200 mg/kg/day group females between Days 1–2. In addition, slight or moderately soft feces
with brown anal fur staining attributed to the color of test substance, were observed in all test substance-administered female groups. Incidences of abnormal breathing sounds on Day 3 in
600 mg/kg/day group female (No. 12504) and 1200 mg/kg/day group Female No. 13506, erected
fur between Days 1–2 in 600 mg/kg/day group Female No. 12501, hunched posture after dose
administration on Day 1 in 300 mg/kg/day group Female No. 11503 were also noted.
Test substance-related decreased activity in 800 mg/kg/day group males between Days 1–2,
abnormal slight or moderately soft feces and brown anal fur staining consistent with the color of
the test substance in all test substance-administered group males between Days 2–3 were observed. In addition, incidences of low carriage in 800 mg/kg/day group Male No. 7005,
abnormal breathing sounds in 800 mg/kg/day group Male No. 7003 and 200 mg/kg/day group
Male No. 5001, soft feces with mucoid material in 400 mg/kg/day group Male No. 6003, and
yellow urogenital fur staining in a 200 mg/kg/day group male, were observed.

Body Weights and Body Weight Gains:
Test substance-related lower body weights were noted in the 400 and 800 mg/kg/day group
males and in the 1200 mg/kg/day group females.
In the 400 and 800 mg/kg/day group males, mean body weight losses of -11.2 g and -29.7 g,
respectively, compared to a mean body weight gain of 2.7 g in the control group between
Days 1–3, were observed. The mean body weight loss was statistically significant at 800 mg/kg/day. On Day 3, the mean body weights in the 400 and 800 mg/kg/day group males
were -5.0% and -13.4% lower, respectively, compared to the control group and corresponded
with the body weight losses. There were no test substance-related effects on body weight in the
200 mg/kg/day group males.
In the 1200 mg/kg/day group females, a statistically significant mean body weight loss of -17.3 g
compared to a mean body weight gain of 0.3 g in the control group between Days 1–3 was noted.
On Day 3, the mean body weight in the 1200 mg/kg/day group females was 10.3% lower than the control group and corresponded with body weight loss. There were no test substance-related
effects on body weights in the 300 and 600 mg/kg/day group females.

Body Temperature:
Test substance-related effects on body temperature were noted in the 800 mg/kg/day group males and in the 1200 mg/kg/day group females.
Test substance-related lower mean body temperatures at 2 and 5 hours post-dose, compared to the control group were observed in the 1200 mg/kg/day group females on Days 1 and 2. The
decreased mean body temperatures were statistically significant at 2 hours post-dose on Day 1
and at 2 and 5 hours postdosing on Day 2. Statistically significant lower mean body temperatures
prior to dose administration on Days 1 and 2, compared to the control group, were considered
incidental.
Test substance-related slightly lower mean body temperatures at 2 and 5 hours postdosing were
observed in the 800 mg/kg/day group males compared to the control group on Day 1. The
differences were not statistically significant but were considered to test substance-related based
on the magnitudes of the decreases compared to the body temperatures before dose
administration.
Any other changes in body temperatures regardless of statistical significance in other test
substance-administered males and females were comparable to changes observed in the control
group and were considered to unrelated to test substance administration.

Bioanalytical Evaluation:
Whole blood samples at 1 hour after dose administration on Day 1 in the 800 mg/kg/day group
males and 1200 mg/kg/day group females were collected for possible future analyses.
The blood samples collected for exposure analyses during the study were not analyzed by the
Sponsor due to clinical observations of brown fur staining in the urogenital and/or anal regions
consistent with the color of the test substance, soft feces, and dose-dependent increases in body
temperature that demonstrated likely evidence of systemic exposure to the test substance.
Furthermore, similar observations of urogenital staining were also noted in a previous study
conducted by the Sponsor (Hannas et al., 2016, Study No. 160038).

Micronucleus Evaluation:
At least 3 dose levels and at least 4000 PCEs per each animal were analyzed to evaluate the
induction of micronuclei after test substance administration. There were at least 5 animals/sex/group for the micronucleus assay evaluation, with the exception of the number of
animals in the 1200 mg/kg/day group females. Due to test substance-related effects on survival
in 2 females and collection of bone marrow from only 3 females, there were less than 5 animals available for scoring the micronuclei; however, the assay was considered scientifically valid because all animals analyzed showed clear negative results without any bone marrow toxicity and the remaining animals allowed for a statistical comparison.
Test substance administration did not result in any statistically significant or dose-related
increases in mean micronucleated polychromatic erythrocytes (%MN-PCE) in both males and
females, compared to concurrent vehicle control group. Means of %MN-PCE in all groups were
below the 95% historical negative control upper limit, with the exception of 800 mg/kg/day
group males (Group 7) that had mean % MN-PCE of 0.29% that was above the upper limit of
95% historical negative control limit at 0.22%. Based on slightly higher %MN-PCE observed in
800 mg/kg/day group males, compared to the control males, 4000 additional PCEs were
evaluated for further investigation. The number of MN-PCEs in the additional cells counted
(Represented as 2nd count), were consistent with the number of MN-PCEs noted, when the initial
4000 cells were counted and did not provide additional clarification. The MN-PCEs observed in
individual animals of 800 mg/kg/day group males were compared to the individual values noted
in the concurrent control group males, and higher mean %MN-PCE of 800 mg/kg/day males was
considered to be due to slightly higher %MN-PCE values of 0.438% and 0.375% noted in Male
No. 7003 and Male No. 7001, respectively. Additionally, Male No. 4003 in the control group
was also observed with a similar higher %MN-PCE value of 0.325%. Based on the lack of a
clear dose response and higher individual values in the control group male, the higher mean %
MN-PCEs in the 800 mg/kg/day males was considered minimal and not biologically relevant.
Therefore, the results for induction of micronuclei were considered as negative.
Test substance administration (4-[3-(1-naphthylamino)propyl]morpholine) did not result in any
notable decreases in the %PCE/TE, which indicated that there was no cytotoxicity in bone
marrow.
Therefore, it was concluded that administration of the test substance for 2 consecutive days did
not result in clastogenic effects or disruption of the mitotic apparatus to induce micronuclei
formation in bone marrow polychromatic erythrocytes of both male and female rats, at dose
levels of 200, 400 and 800 mg/kg/day in males and at 300, 600 and 1200 mg/kg/day for females.
Thus, the test substance was considered not to be genotoxic in this in vivo micronucleus
evaluation.

Summary of Body Weights: Range-Finding Phase
Bodyweight (g)		Day(s) Relative to start date					Day(s) Relative to start date
Sex: Male		1	2	3	Sex: Female		1	2	3
200 mg/kg/day: Group 1	MEAN	186.0	183.3	189.3	200 mg/kg/day: Group 1	MEAN	174.7	166.0	164.0
	SD	23.1	23.3	21.7		SD	4.6	2.6	3.6
	N	3	3	3		N	3	3	3
400 mg/kg/day: Group 2	MEAN	173.0	159.0	161.7	400 mg/kg/day: Group 2	MEAN	172.3	166.7	168.3
	SD	15.5	9.6	14.2		SD	6.7	8.7	8.1
	N	3	3	3		N	3	3	3
800 mg/kg/day: Group 3	MEAN	188.7	176.7	179.3	800 mg/kg/day: Group 3	MEAN	170.7	157.0	156.7
	SD	20.5	25.0	21.7		SD	7.6	6.6	10.1
	N	3	3	3		N	3	3	3
1200 mg/kg/day: Group 9	MEAN	333.3	301.5	293.5	1200 mg/kg/day: Group 9	MEAN	234.7	218.3	211.0
	SD	6.1	3.5	2.1		SD	7.8	8.3	13.5
	N	3	2	2		N	3	3	3
2000 mg/kg/day: Group 10	MEAN	327.3	309.0	306.0	2000 mg/kg/day: Group 10	MEAN	222.7	201.0	207.5
	SD	6.4				SD	26.1	18.7	7.8
	N	3	1	1		N	3	3	2

Summary of bodyweight gains (g): Range finding phase
Bodyweight gain (Interval)		Day(s) Relative to start date					Day(s) Relative to start date
Sex: Male		1 to 2	2 to 3	1 to 3	Sex: Female		1 to 2	2 to 3	1 to 3
200 mg/kg/day: Group 1	MEAN	-2.7	6.0	3.3	200 mg/kg/day: Group 1	MEAN	-8.2	-2.0	-10.7
	SD	2.1	2.0	1.5		SD	2.1	2.0	3.2
	N	3	3	3		N	3	3	3
400 mg/kg/day: Group 2	MEAN	-14.0	2.7	-11.3	400 mg/kg/day: Group 2	MEAN	-5.7	1.7	-4.0
	SD	9.5	13.1	7.1		SD	5.5	2.5	7.0
	N	3	3	3		N	3	3	3
800 mg/kg/day: Group 3	MEAN	-12.0	2.7	-9.3	800 mg/kg/day: Group 3	MEAN	-13.7	-0.3	-14.0
	SD	6.6	8.6	3.1		SD	7.5	3.5	9.6
	N	3	3	3		N	3	3	3
1200 mg/kg/day: Group 9	MEAN	-34.5	-8.0	-42.5	1200 mg/kg/day: Group 9	MEAN	-16.3	-7.3	-23.7
	SD	2.1	1.4	3.5		SD	4.0	5.7	9.2
	N	2	2	2		N	3	3	3
2000 mg/kg/day: Group 10	MEAN	-22.0	-3.0	-25.0	2000 mg/kg/day: Group 10	MEAN	-21.7	-1.0	-27.5
	SD	-	-	-		SD	8.5	11.3	13.4
	N	1	1	1		N	3	2	2

Summary of Body Weights: Main Study
Bodyweight (g)		Day(s) Relative to start date					Day(s) Relative to start date
Sex: Male		1	2	3	Sex: Female		1	2	3
0 mg/kg/day: Group 4	MEAN	297.7	295.7	300.3	0 mg/kg/day: Group 4	MEAN	195.5	192.5	195.8
	SD	22.9	24.5	29.2		SD	14.1	11.5	14.6
	N	6	6	6		N	6	6	6
200 mg/kg/day: Group 5	MEAN	291.5	286.2	290.7	300 mg/kg/day: Group 11	MEAN	192.5	189.5	187.7
	SD	27.7	33.8	29.2		SD	17.2	12.6	16.6
	N	6	6	6		N	6	6	6
	%Diff	-2.1	-3.2	-3.2		%Diff	-1.5	-1.6	-4.2
400 mg/kg/day: Group 6	MEAN	296.3	284.3	285.2	600 mg/kg/day: Group 12	MEAN	195.0	188.8	192.3
	SD	22.7	30.4	33.7		SD	14.9	14.5	14.8
	N	6	6	6		N	6	6	6
	%Diff	-0.4	-3.8	-5.0		%Diff	-0.3	-1.9	-1.8
800 mg/kg/day: Group 7	MEAN	289.8	269.0	260.2	1200 mg/kg/day: Group 13	MEAN	192.2	186.0	175.8
	SD	16.3	14.0	15.0		SD	192.2	186.0	175.8
	N	6	6	6		N	6	5	4
	%Diff	-2.6	-9.0	-13.4		%Diff	-1.7	-3.4	-10.3
Summary of bodyweight gains (g): Main study
Bodyweight gain (Interval)		Day(s) Relative to start date					Day(s) Relative to start date
Sex: Male		1 to 2	2 to 3	1 to 3	Sex: Female		1 to 2	2 to 3	1 to 3
0 mg/kg/day: Group 4	MEAN	-2.0	4.7	2.7	0 mg/kg/day: Group 4	MEAN	-3.0	3.3	0.3
	SD	7.6	7.4	10.5		SD	7.2	5.4	5.3
	N	6	6	6		N	6	6	6
200 mg/kg/day: Group 5	MEAN	-5.3	4.5	-0.8	300 mg/kg/day: Group 11	MEAN	-3.0	-1.8	-4.8
	SD	7.5	6.4	3.6		SD	7.3	7.8	11.7
	N	6	6	6		N	6	6	6
400 mg/kg/day: Group 6	MEAN	-12.0	0.8	-11.2	600 mg/kg/day: Group 12	MEAN	-6.2	3.5	-2.7
	SD	9.7	6.7	13.5		SD	7.8	4.3	5.9
	N	6	6	6		N	6	6	6
800 mg/kg/day: Group 7	MEAN	-20.8	-8.8*	-29.7**	1200 mg/kg/day: Group 13	MEAN	-9.6	-9.5**	-17.3*
	SD	9.9**	8.2	13.5		SD	5.8	5.3	8.5
	N	6	6	6		N	5	4	4

Summary of Micronucleus Results
Sampling Time - Day 3 (18-24 Hours post last dose)
Male
Group 4 - Vehicle
Group 6 - Test Substance 400 mg/kg/day
Group 5 - Test Substance 200 mg/kg/day
Group 7 - Test Substance 800 mg/kg/day
Group 8 - Positive Control 20/mg/kg/day
					% MN-PCE^a
Group	Number of Animals	Mean %PCE/TE	%RPCE/TE	Mean Incidence MN-PCE	Mean	±	SD
4	5	42.1	100.0	15.4	0.19	±	0.08
5	5	57.2	135.9	14.8	0.19	±	0.10
6	5	50.8	120.8	12.6	0.16	±	0.04
7	5	48.2	114.6	22.8	0.29	±	0.11
8	3	53.6	127.5	109.7	2.74 A	±	1.30
A = Significant increase from control group (Group 4) value: A - P ≤ 0.05 B - P ≤ 0.01 (Wilcoxon)
Sampling Time - Day 3 (18-24 Hours post last dose)
Female
Group 4 - Vehicle
Group 11 - Test Substance 300 mg/kg/day
Group 12 - Test Substance 600 mg/kg/day
Group 13 - Test Substance 1200 mg/kg/day
					% MN-PCE^a
Group	Number of Animals	Mean %PCE/TE	%RPCE/TE	Mean Incidence MN-PCE	Mean	±	SD
4	5	56.4	100.0	7.4	0.19	±	0.04
11	5	54.0	95.6	7.0	0.18	±	0.08
12	5	52.1	92.3	4.2	0.11	±	0.06
13	3	49.0	86.9	6.0	0.15	±	0.03
a - statistical analysis performed on %MN-PCE

Micronucleus test in bone marrow of male rats group results
Studies Performed Between Mar 2015 - Aug 2021
Historical negative control data
	Frequency of MN-PCE^a	% MN-PCE	% PCE/TE
Mean ± SD	5.3 ± 1.8	0.13 ± 0.04	54.6 ± 9.8
Range (min-max)	1.8 - 11.4	0.05 - 0.29	27.2 - 78.0
Number of experiments	108	108	108
95% Control Limits	1.8-8.8	0.05 - 0.22
Historical positive control data^b
	Frequency of MN-PCE^a	% MN-PCE	% PCE/TE
Mean ± SD	80.0 ± 28.5	2.01 ± 0.71	37.9 ± 8.7
Range (min-max)	31.0 - 214.7	0.78 - 5.37	8.2 - 59.1
Number of experiments	115	115	115
a - Number of MN-PCE observed per 4000 PCE examined
b - Rats were treated using CP (20 mg/kg/day) or CP and EMS (200 or 241 mg/kg/day
Micronucleus test in bone marrow of female rats group results
Studies Performed Between Mar 2015 - Aug 2021
Historical negative control data
	Frequency of MN-PCE^a	% MN-PCE	% PCE/TE
Mean ± SD	4.8 ± 1.8	0.12 ± 0.05	47.7 ± 6.8
Range (min-max)	1.5 - 9.6	0.04 - 0.24	28.7 - 62.9
Number of experiments	51	51	51
95% Control Limits	1.2 - 8.4	0.03 - 0.21
a - Number of MN-PCE observed per 4000 PCE examined

Conclusions:

Administration of 4-[3-(1-naphthylamino)propyl]morpholine by once daily oral gavage for 2 consecutive days to Crl:CD(SD) rats resulted in mortality at dose levels ≥ 1200 mg/kg/day in males and at 2000 mg/kg/day in females in the range-finding phase. In the definitive phase, effects on survival were also observed at 1200 mg/kg/day in females. Based on effects on survival in both phases, the maximum tolerated dose (MTD) level was considered to be 800 mg/kg/day for both males and females. Test substance-related lower body weights at ≥ 400 mg/kg/day in males and 1200 mg/kg/day females, decreases in body temperatures were observed at ≥ 800 mg/kg/day in males and females. There was no clear evidence of micronuclei induction in PCEs in bone morrow or bone marrow cytotoxicity in males or females. Based on these results, administration of 4-[3- (1 naphthylamino)propyl]morpholine at dose levels up to 800 mg/kg/day in males and 1200 mg/kg/day in females for 2 days by daily oral gavage, did not show a clear evidence of micronuclei induction genotoxicity in rats, but a dose level above 800 mg/kg/day was considered to exceed the maximum tolerated dose.

Executive summary:

The objective of this study was to assess the potential toxicity of the test substance to induce micronuclei in polychromatic erythrocytes in rat bone marrow following 2 consecutive days of
treatment when administered by oral gavage.

The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, body temperatures, and micronucleus evaluation (definitive
phase only), and macroscopic examinations (gross necropsy for animals found dead).

Range-Finding Phase:
Test substance-related effects on survival were observed at ≥ 1200 mg/kg/day in males and at 2000 mg/kg/day in females. Two males and 1 female at 2000 mg/kg/day and 1 male at 1200 mg/kg/day were found dead prior the scheduled euthanasia. Clinical observations noted for these animals prior to being found dead included low carriage, slight incoordination, hunched posture, decreased activity, slightly soft feces, brown urogenital staining consistent with the color of the test substance, eyes partly closed, erected fur, weak, and/or lying on side.
All other animals survived to the scheduled euthanasia.
Test substance-related clinical observations were observed at ≥ 200 mg/kg/day in females and at ≥ 400 mg/kg/day in males. At 2000 mg/kg/day, clinical observations included decreased activity,
hunched posture, slightly soft feces, brown staining of the fur in the urogenital area, and low carriage in both males and females, slight incoordination, and erected fur in females. At 1200 mg/kg/day, observations included decreased activity, low carriage and hunched posture in both males and females, slight incoordination in males, and skin pallor, slightly soft feces and
brown urogenital fur staining as consistent with the color of the test substance in females. At 800 mg/kg/day observations included ploughing behavior in males and females, brown staining and wet fur in the urogenital region as consistent with the color of the test substance, and slightly soft feces in females. At 400 mg/kg/day ploughing behavior in males and females, and slightly soft feces in males were noted. At 200 mg/kg/day, observations were limited to ploughing behavior in females.
Test substance-related body weight losses were observed between Days 1–3, in males at ≥ 400 mg/kg/day and ≥ 200 mg/kg/day in females. On Day 3, the mean body weights in the 400, 800, 1200, and 2000 mg/kg/day group males were decreased by 6.5%, 5.0%, 11.9%, and 6.5%, respectively, compared to Day 1 mean body weights. On Day 3, the mean body weights in the 200, 400, 800, 1200, and 2000 mg/kg/day group females were decreased by 6.1%, 2.3%, 8.2%, 10.1%, and 6.8%, respectively, compared to Day 1 mean body weights.
Test substance-related effects on body temperature were noted in the ≥ 200 mg/kg/day group males and females, when compared to their mean body temperatures before dose administration on Days 1 and 2. A qualitative comparison of the decreased body temperatures compared to predose body temperatures showed a dose-dependent trend with higher magnitudes of decreased body temperatures observed at 1200 and 2000 mg/kg/day in males and at 800, 1200, and 2000 mg/kg/day in females.

Definitive Phase:
Test substance-related effects on survival were observed in 1200 mg/kg/day group females. Two females were found dead: 1 on Day 2 and 1 on Day 3. Clinical observations in these animals before being found dead during the study included decreased activity, low carriage, moderate incoordination, abnormal gait, moderate amount of soft feces, and/or brown fur staining on the anal region.

All other animals survived to the scheduled euthanasia.
Test substance-related clinical observations were noted in all test substance-administered males and females. Test substance-related observations included hunched posture in all test substance-administered female groups, low carriage, moderate incoordination, and decreased activity in the 600 and 1200 mg/kg/day group females between Days 1–2. Slight or moderately soft feces with brown anal fur staining attributed to the color of test substance were observed in all test substance-administered female groups. In addition, single incidences of abnormal breathing
sounds in a 600 mg/kg/day female and a 1200 mg/kg/day female on Day 3, erected fur in a 600 mg/kg/day female between Days 1–2, hunched posture after dose administration in a 300 mg/kg/day female on Day 1 were observed. In males, test substance-related decreased activity was observed in 800 mg/kg/day group males between Days 1–2. Abnormal slight or moderately soft feces and brown anal fur staining attributed to the color of test substance was observed in all test substance administered group males between Days 2–3. Single incidences of low carriage in an 800 mg/kg/day group male, abnormal breathing sounds in an 800 mg/kg/day group male and a 200 mg/kg/day group male, soft feces with mucoid material in a 400 mg/kg/day group male, and yellow urogenital fur staining consistent with the color of the
test substance in a 200 mg/kg/day group male, were also observed.

Test substance-related lower body weights were observed in the 400 and 800 mg/kg/day group males and the 1200 mg/kg/day group females. Mean body weights on Day 3 were 5.0% and 13.4% lower in the 400 and 800 mg/kg/day group males, respectively, compared to the control group. In the 1200 mg/kg/day group females, mean body weights were 10.3% lower on Day 3 when compared to the control group. Lower mean body weights in both males and females were associated with body weight losses observed between Days 1–3.
Test substance-related effects on body temperature were noted in 800 mg/kg/day group males and 1200 mg/kg/day group females. In the 1200 mg/kg/day group females, test substance-related
lower mean body temperatures at 2 and 5 hours postdose were noted compared to the control group on Days 1 and 2. In the 800 mg/kg/day group males, test substance-related slightly lower
mean body temperatures at 2 and 5 hours postdosing were noted compared to the control group on Day 1.
Test substance administration in males and females did not result in any statistically significant increases in the means of micronucleated polychromatic erythrocytes (%MN-PCE), compared to the vehicle control group. Group means of %MN-PCE in test substance-administered groups were within the 95% limits of the historical negative control distribution in all test substance-administered groups, with the exception of 800 mg/kg/day group males that had mean %MN-PCE above 95% historical negative control data but within the historical range. Thus, an
additional 4000 cells for each animal were analyzed for induction of micronuclei and the overall results were considered as negative as the increases were minimal compared to the individual
values noted in concurrent control and thus were considered biologically not relevant. There were no substantial decreases in the proportion of %PCE/TE, indicating that there was no bone
marrow cytotoxicity, after the administration of test substance.
Administration of 4-[3-(1-naphthylamino)propyl]morpholine by once daily oral gavage for 2 consecutive days to Crl:CD(SD) rats resulted in mortality at dose levels ≥ 1200 mg/kg/day in
males and at 2000 mg/kg/day in females in the range-finding phase. In the definitive phase, effects on survival were also observed at 1200 mg/kg/day in females. Based on effects on survival in both phases, the maximum tolerated dose (MTD) level was considered to be 800 mg/kg/day for both males and females. Test substance-related decreases in body temperatures were observed at ≥ 800 mg/kg/day in males and females. Also, significant changes in body weight were noted at 800 mg/kg/day in males and 1200 mg/kg/day in females. There was no clear evidence of micronuclei induction in PCEs in bone morrow or bone marrow cytotoxicity in males or females. Based on these results, administration of 4-[3-(1 naphthylamino)propyl]morpholine at dose levels up to 800 mg/kg/day in males and 1200 mg/kg/day in females for 2 days by daily oral gavage, did not show a clear evidence of micronuclei induction genotoxicity in rats, but a dose level above 800 mg/kg/day were considered to exceed the maximum tolerated dose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro Studies:

Bacterial Mutagenicity:

 

The potential of 4-(3-(1-Naphthylamino)propyl)morpholine to induce reverse mutations in Salmonella typhimurium strains TA1537, TA1535, TA98 and TA100 anda tryptophan deficient strain, Escherichia coli WP2uvrA (pKM101) was evaluated in the bacterial reverse mutation test using the pre-incubation method.

4-(3-(1-Naphthylamino)propyl)morpholinewas tested in the absence and presence of metabolic activation using dimethyl sulfoxide (DMSO) as the solvent. In the Initial Toxicity Mutation Assay, bacterial cultures were exposed to 4-(3-(1-Naphthylamino)propyl)morpholine at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (two plates/concentration). Precipitation was not observed up to the concentration of 5000 µg/plate. Complete inhibition of background lawn was observed at the tested concentrations of 1500 and 5000 µg/plate in all the tester strains both in absence and presence of metabolic activation (5% v/vS9 mix). Partial inhibition of background lawn was observed at tested concentration of 500 µg/plate in all the tester strains both in absence and presence of metabolic activation. Normal background lawn pattern without reduction in number of revertant colonies was observed up to the tested concentration of 150 µg/plate in all the tester strains in both absence and presence of metabolic activation (5% v/vS9 mix). No increase in the number of revertant colonies was observed in any of the tester strains with the test item at any of the tested concentrations in the absence or presence of metabolic activation when compared with the concurrent negative control. 

 

In the Confirmatory Mutation Assay, bacterial cultures were exposed to 4-(3-(1-Naphthylamino)propyl)morpholine at concentrations of 31.25, 62.5, 125, 250, 500 and 1000 µg/plate (three plates/concentration) both in the absence and presence of metabolic activation (10% v/vS9 mix). Precipitation was not observed up to the concentration of 1000 µg/plate. Partial inhibition of background lawn was observed at the tested concentrations of 500 and 1000 µg/plate in all the tester strains both in absence and presence of metabolic activation. No inhibition of background lawn was observed up to the tested concentration of 250 µg/plate in all the tester strains in both absence and presence of metabolic activation. No increase in the number of revertant colonies was observed in any of the tester strains with the test item at any of the tested concentrations in the absence or presence of metabolic activation when compared with the concurrent negative control.

 

All the values for the negative controls were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

 

The 4-(3-(1-Naphthylamino)propyl)morpholine stock concentrations 312.5, 625, 1250, 2500, 5000 and    10000 µg/mL were found to be within acceptable range of ± 15% of nominal concentrations during the Confirmatory Mutation Assay. The 0 hour concentrations of 4-(3-(1-Naphthylamino)propyl)morpholine in the dose formulation were found to be 108%, 106%, 107%, 103%, 97.4%, 96.9% of the nominal concentrations of dose level T1 (312.5 µg/mL), T2 (625 µg/mL), T3 (1250 µg/mL), T4 (2500 µg/mL), T5 (5000 µg/mL) and T6 (10000 µg/mL), respectively. The concentrations of 4-(3-(1-Naphthylamino)propyl)morpholine in the dose formulation after 4 hours were found to be 99.3% and 99.1% of the 0 hour concentrations of dose level T1 (312.5 µg/mL) and T6 (10000 µg/mL), respectively. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient.

 

All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, 4-(3-(1-Naphthylamino)propyl)morpholine is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2uvrA (pKM101).

 

In vitro Clastogenicity (mammalian cells)

 

4-(3-(1-naphthylamino)propyl)morpholine (N-1-Naphthalenyl-4-morpholinepropanamine) was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes.

Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 677.8 μg 4-(3-(1-naphthylamino)propyl)morpholine per ml of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium. The analytically determined concentrations of 4-(3-(1-naphthylamino)propyl)morpholine in the dose preparations ranged from 93.0 to 100.7% of the targeted values. The duration of treatment was 4 hours without and with S9 and 24 hours without S9. Selection of concentrations for the determination of the incidence of chromosomal aberrations was based upon cytotoxicity of the test material. In this study cultures treated for 4 hours with targeted concentrations of 0 (vehicle control), 10.6, 42.4, and 169.5 μg/ml in the absence of S9, 0 (vehicle control), 2.6, 5.3, and 10.6 μg/ml in the presence of S9, and cultures treated for 24 hours with targeted concentrations of 0 (vehicle control), 5.3, 21.2, and 42.4 μg/ml were analyzed for chromosomal aberration frequency.

There were no significant increases in the frequency of cells with aberrations administered 4-(3-(1-naphthylamino)propyl)morpholine in the absence of S9 activation. There was a significant increase in the frequency of cells with aberrations in the presence of S9, with 0.0% in the vehicle control and 2.7, 3.7, and 4.4% in the treatment concentrations (i.e., 2.6, 5.3, and 10.6 μg/ml), respectively. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of aberrant cells. The vehicle and positive controls in all assays were within the acceptable historical ranges and fulfilled the requirements for a valid assay.

Based upon these results, 4-(3-(1-naphthylamino)propyl)morpholine was considered to be positive for clastogenicity in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

 

In vivo Clastogenicity:

 

Due to the positive finding in the in vitro clastogenicity assay an in vivo micronucleus assay was conducted in order to assess the in vivo clastogenic potential of this substance. Administration of 4-[3-(1-naphthylamino)propyl]morpholine by once daily oral gavage for 2 consecutive days to Crl:CD(SD) rats resulted in mortality at dose levels ≥ 1200 mg/kg/day in males and at 2000 mg/kg/day in females in the range-finding phase. In the definitive phase, effects on survival were also observed at 1200 mg/kg/day in females. Based on effects on survival in both phases, the maximum tolerated dose (MTD) level was considered to be 800 mg/kg/day for both males and females. Test substance-related lower body weights at ≥ 400 mg/kg/day in males and 1200 mg/kg/day females, decreases in body temperatures were observed at ≥ 800 mg/kg/day in males and females. There was no clear evidence of micronuclei induction in PCEs in bone morrow or bone marrow cytotoxicity in males or females. Based on these results, administration of 4-[3- (1 naphthylamino)propyl]morpholine at dose levels up to 800 mg/kg/day in males and 1200 mg/kg/day in females for 2 days by daily oral gavage, did not show a clear evidence of micronuclei induction genotoxicity in rats, but a dose level above 800 mg/kg/day was considered to exceed the maximum tolerated dose.

Justification for classification or non-classification

Based on the negative in vivo clastogenicity assay and negative in vitro Ames assay the substance does not meet the criteria for classification in accordance with Regulation EC 1272/2008.