1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A Bacterial Reverse Mutation Assay and an in vitro mammalian chromosomal aberration assay were carried out on 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether in accordance with OECD guidelines 471 and 473 respectively.

The bacterial reverse mutation assay was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted. The in vitro mammalian chromosomal aberration assay carried out on 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether was concluded to be negative for the induction of structural chromosomal aberrations and positive for the induction of numerical chromosomal aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosomal aberration assay using CHO cells.

For purposes outside of the EU and as a result of the positive results (numerical aberrations) within the chromosome aberration test the test substance, 1,3 -Benzenedimethanamine, reaction products with glycidyl tolyl ether, was further tested for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in rat bone marrow.

Corn Oil was selected as the vehicle. Test and/or control substance formulations were administered once per day on two consecutive days at a dose volume of 10 mL/kg by oral gavage.

In the dose range-finding assay (DRF), the maximum dose tested was 2000 mg/kg/day. The dose levels tested were 500, 1000, and 2000 mg/kg/day in 3 of animals/sex. Based upon the results, the high dose for the

definitive assay was 500 mg/kg/day, which was estimated to be the maximum tolerated dose (MTD).

The definitive assay dose levels tested were 125, 250, and 500 mg/kg/day.

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 administration of 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether at doses up to and including a dose of 125, 250, and 500 mg/kg/day was concluded to be

negative in the Micronucleus assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 September 2016 to 26 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline 471, updated and adopted 21 July 1997; ISO/IEC 17025:2005

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Remarks:

Refer to main study report

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: WA1508 (provided by Sponsor)STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature, protected from light- Solubility of the test substance in the solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.TREATMENT OF TEST MATERIAL PRIOR TO TESTING- Final dilution of a dissolved solid, stock liquid or gel: To achieve a solution, the most concentrated dilution was vortexed for two minutes in the mutagenicity assay.

Target gene:

The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. These strains contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain from the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene, which codes for a protein of the DNA nucleotide excision repair system, resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chi) and biotin (bio) genes (bacteria require biotin for growth). Tester strains TA98 and TA100 contain the R-factor plasmid, pKM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non-R-factor parent strains. pKM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system, which is normally present in these organisms. The tester strain Escherichia coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens, which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.

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:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate.In the mutagenicity assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO for the test substance; all positive controls were diluted in dimethyl sulfoxide (DMSO) except for sodium azide, which was diluted in sterile water- Justification for choice of solvent/vehicle:DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Exposure duration: 48 to 72 hoursNUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 3 in the mutagenicity assayNUMBER OF CELLS EVALUATED: >/= 0.3 x 10^8 cells/plateDETERMINATION OF CYTOTOXICITY- A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction inthe mean number of revertants per plate as compared to the mean vehicle control value. This reductionmust be accompanied by an abrupt dose dependent drop in the revertant count. (2) At least a moderatereduction in the background lawn (background code 3, 4 or 5).

Evaluation criteria:

The revertant colony numbers were determined for each plate (counted either manually or by automatic colony counter). The mean and standard deviation of the number of revertants per plate were calculated and reported.For the test substance 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 substance as specified below:Strains TA1535 and TA1537Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.Strains TA98, TA100 and WP2 uvrAData sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.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:

According to the test guidelines, the biological relevance of the results is the criterion for the interpretation of the results, and a statistical evaluation of the results is not regarded as necessary.

Key result

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the preliminary toxicity assay, toxicity was observed beginning at concentrations from 66.7 to 3333 ug per plate. In the mutagenicity assay, toxicity was observed beginning at 150, 500 or 1500 ug per plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: No precipitate was observed.HISTORICAL CONTROL DATA with ranges, means and standard deviation and confidence interval (e.g. 95%)See main study report appendix

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether 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. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Executive summary:

All criteria for a valid study were met as described in the protocol.

The test substance, 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. No precipitate was observed. Toxicity was observed beginning at concentrations from 66.7 to 3333 μg per plate. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate.

In the mutagenicity assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity was observed beginning at 150, 500 or 1500 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether was negative for the ability to induce reverse mutations at selected loci of several strains ofSalmonellatyphimurium and at the tryptophan locus ofEscherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 September 2016 to 23 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline 473, updated and adopted 26 September 2014

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Remarks:

Refer to main study report

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: WA1508STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature, protected from light

Target gene:

chromosomes

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the doses tested were 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/mL.In the chromosome aberration assay, the doses tested were as follows:Non-activated 4-hour treatment --> 2.5, 5, 10, 12.5, 15, 17.5, 20, 25 and 30 ug/mLS9-activated 4-hour treatment --> 2.5, 5, 6, 7.5, 8, 9, 10, 11, 12.5 and 15 ug/mLNon-activated 20-hour treatment --> 15, 30, 40, 50, 60, 70, 80, 90, 100 and 125 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO) - Justification for choice of solvent/vehicle: DMSO was used as the vehicle 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 DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in mediumDURATION- Exposure duration: CHO cells were exposed to the test and control articles for 4 and 20 hours without S9 and for 4 hours with S9, and rinsed. Cells were harvested 20 hours (±30 minutes) after initiation of treatment, which corresponds to 1.5 normal cell cycles.STAIN (for cytogenetic assays): GiemsaNUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 2 in the chromosome aberration assayNUMBER OF CELLS EVALUATED: a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture), whenever possibleDETERMINATION OF CYTOTOXICITY- Method: Cell growth index (measurement of RICC)OTHER EXAMINATIONS:- Determination of polyploidy: yes- Determination of endoreplication: yes

Evaluation criteria:

Toxicity induced by treatment was based upon inhibition of cell growth and was reported for the cytotoxicity and chromosome aberration portions of the study. 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 are 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.A test article was considered positive if it induced a statistically significant and dose dependent increase in the frequency of aberrant metaphases (p

Statistics:

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

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

other: Negative for the induction of structural chromosomal aberrations and positive for the induction of numerical chromosomal aberrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: Acceptable- Effects of osmolality: Acceptable- Precipitation: In the preliminary toxicity assay, at the conclusion of the treatment period, visible precipitate was observed at doses greater than 500 µg/mL in all three exposure groups.- Definition of acceptable cells for analysis: Metaphase spreads containing 20 ± 2 centromeresHISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)- Positive historical control data: Refer to main study report- Negative (solvent/vehicle) historical control data: Refer to main study reportADDITIONAL INFORMATION ON CYTOTOXICITY:- Measurement of cytotoxicity used: RICC- Other observations when applicable: Monolayer confluency and mitotic inhibition

Conclusions:

Under the conditions of the assay described in this report, 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether was concluded to be negative for the induction of structural chromosomal aberrations and positive for the induction of numerical chromosomal aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosomal aberration assay using CHO cells.

Executive summary:

The test substance,1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether, 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 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. Dimethyl sulfoxide (DMSO)was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 µg/mL, which was the limit dose for this assay. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 15 µg/mL in the non-activated 4 and 20-hour exposure groups; and at doses ≥ 150 µg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 500 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the chromosomal aberration assay ranged from 2.5 to 30 µg/mL for the non-activated 4-hour exposure group; from 15 to 125 µg/mL for the S9-activated 4-hour exposure group; and from 2.5 to 15 µg/mL for the non-activated 20-hour exposure group.

In the chromosomal aberration assay, cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control), was observed at doses ≥ 15 µg/mL in the non-activated 4-hour exposure group; at doses ≥ 40 µg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 10 µg/mL in the non-activated 20-hour exposure group. The doses selected for evaluation of chromosomal aberrations were 10, 12.5, and 15 µg/mL for the non-activated 4-hour exposure group; 15, 30, and 40 µg/mL for the S9-activated 4-hour exposure group; and 2.5, 6, and 10 µg/mL for the non-activated 20-hour exposure group.

 

No significant or dose-dependent increases in structural chromosomal aberrations were observed at any dose in any of the treatment groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the non-activated 4-hour exposure group, statistically significant increases (10.3% and 11.7%) in numerical aberrations was observed at doses 12.5 and 15 µg/mL, respectively (p ≤ 0.01; Fisher’s Exact). The Cochran-Armitage test was also positive for a dose response (p ≤ 0.05).

In the S9-activated 4-hour exposure group, statistically significant increases (16.3% and 12.7%) in numerical aberrations was observed at doses 30 and 40 µg/mL, respectively (p ≤ 0.01; Fisher’s Exact). The Cochran-Armitage test was also positive for a dose response (p ≤ 0.05).

In the non-activated 20-hour exposure group, a statistically significant increase (5.0%) in numerical aberrations was observed at 10 µg/mL (p ≤ 0.05; Fisher’s Exact). However, the Cochran-Armitage test was negative for dose-response (p > 0.05).

These results indicate that1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether was negative for the induction of structural chromosomal aberrations and positive for the induction of numerical chromosomal aberrations in the presence and absence of the exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo

An in vivo mammalian somatic cell study using male Sprague-Dawley rats was used to test 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether. The testing was carried out according to OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test) 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:

22 June 2017 to 5 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline 474 (OECD, 2016) and ISO/IEC 17025:2005 (ISO/IEC, 2005)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

The Study Director failed to issue an amendment on time. Certain gavage checks were not documented in the study book. Instead, typed in an email to the Study director. The appropriate actions did occur. Neither deviation had an adverse impact on the study

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mammalian Erythrocyte Micronucleus Assay in Rats

Specific details on test material used for the study:

Source and lot/batch No.of test material: WA1508
Corn oil was the vehicle of choice based on the solubility of the test substance, and compatibility with the test system.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature, protected from light
- Solubility of the test substance in the solvent/vehicle: The test substance was soluble in corn oil at a
maximum concentration of approximately 200 mg/mL in the solubility test conducted at BioReliance.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The formulation was vortexed (in the DRF only), then stirred
magnetically for 15-37 minutes until uniform.

Species:

rat

Strain:

Sprague-Dawley

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:

- Source: Envigo RMS, Inc., Frederick, MD
- Age at study initiation: 6 weeks
- Weight at study initiation:
DRF assay: Males -> 172.2 - 191.8 grams; Females -> 142.9 - 156.7 grams
Definitive mutagenicity assay: Males -> 162.7 - 182.7 grams
- Assigned to test groups randomly: [no/yes, under following basis: ] yes
- Fasting period before study: no
- Housing: Animals of the same sex were housed up to five per Micro-Barrier cage.
- Diet (e.g. ad libitum): A certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein
Rodent Diet was provided ad libitum.
- Water (e.g. ad libitum): Animals had free access to tap water, which met US EPA drinking water standar
ds.
- Acclimation period: Animals were acclimated for 5 days in the DRF assay and for 6 days in the Definit
ive assay.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 +/-3 degrees F
- 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
IN-LIFE DATES:
DRF assay - From: 23 Jun 2017 To: 24 Jun 2017
Definitive assay - From: 09 Aug 2017 To: 11 Aug 2017

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used:
For test article: Corn Oil
For positive control, if applicable: Scoring positive control slides, generated in a separate study, were
included to verify scoring. In that study, the positive control was prepared in deionized water.
- Justification for choice of solvent/vehicle: Corn oil was the vehicle of choice based on the solubility of
the test substance and compatibility with the test system.
- Concentration of test material in vehicle: nominally 200 mg/mL at the top dose in the DRF and nomi
nally 50 mg/mL at the top dose in the Definitive
- Lot/batch no. of test article vehicle (if required): MKBW9504V (DRF); MKCC0462 (Definitive)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: A suitable sized amber glass vial with a PTFE stir bar, co
ntaining a pre-weighed amount of test substance was calibrated to the final batch size. The formulation
was QS’ed to the final volume. The formulation was vortexed (in the DRF only); then stirred magneticall
y for 15-37 minutes until uniform. The final formulation was maintained at room temperature until use the
same day.

Duration of treatment / exposure:

All dose formulations were administered once daily for two consecutive days.

Frequency of treatment:

All dose formulations were administered once daily for two consecutive days.

Dose / conc.:

125 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose:

DRF: 3
Definitive: 5 in Groups 1-3, 5+2 in Group 4 (2 additional animals were added to cover in the event of
mortality. Only 5 animals were used for micronucleus evaluation, and any additional animals were e
uthanized without further examination.)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate (CP)
Scoring positive control slides (fixed and unstained), generated from BioReliance Study No. AE45KT.125
M012.BTL were included to verify scoring. These slides were generated from male rats treated once w
ith cyclophosphamide monohydrate (CP) at 40 mg/kg, and the bone marrow harvested 24 hours after tre
atment.
- Route of administration: oral gavage

Tissues and cell types examined:

Femoral bone marrow was collected at approximately 24 hours after the final dose and evaluated by fl
uorescent microscopy. At least 4000 PCEs/animal were scored for the presence of micronuclei (MnPCE
s), whenever possible. 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.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In the dose range finding assay, 500, 1000 and 2000 mg/kg/day were selected as the dose levels. Three
animals/sex were tested up to the limit dose of 2000 mg/kg/day.
In the definitive assay, 125, 250 and 500 mg/kg/day were selected as the dose levels based on the resu
lts of the dose range finding assay.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): All dose formulations
were administered once per day on two consecutive days at a dose volume of 10 mL/kg by oral gavage.
In the definitive assay, femoral bone marrow was collected at approximately 24 hours after the final dose
for microscopic evaluation.
DETAILS OF SLIDE PREPARATION: Approximately 24 hours after the final dose, animals were euth
anized 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 bovi
ne serum with the pellet. Cells were resuspended 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 slides (including at least five positive control slides) was stained wi
th acridine orange for microscopic evaluation. Stained slides were discarded prior to report finalization.

The other set of slides was kept as backup and will be archived at report finalization. Each slide was ide
ntified by harvest date, study number and animal number. Slides were coded using a random number
table by an individual not involved in the scoring process.
METHOD OF ANALYSIS: microscopic evaluation

Evaluation criteria:

A test article was considered to have induced a positive response if:
a) at least one of the test article doses exhibited a statistically significant increase when compared with
the concurrent vehicle control (pb) when multiple doses were examined at a particular sampling time, the increase was dose-related (p0.01), and
c) results of the group mean or of the individual animals in at least one group were outside the 95% cont
rol limit of the historical vehicle control data.
A test article 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 article
(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 t
reatment 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 articl
e groups at the respective sampling times were compared used Levene's test (significance level of p= 0.05). If the variation between groups was found not to be significant, a parametric one-way ANOVA
was performed followed by a Dunnett's post-hoc analysis to compare each dose group to the concurrent
vehicle control group. If Levene's test indicated heterogeneous group variances (parametric statistical method (Kruskal-Wallis and/or Mann-Whitney) was used in the evaluation of data.
A linear regression analysis was conducted to assess dose responsiveness in the test article treated gro
ups (pA 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:

yes

Remarks:

Clinical signs of piloerection were observed at the high dose of 500 mg/kg/day in the Definitive assay.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000 and 2000 mg/kg/day
- Solubility: The test substance was soluble in corn oil at a maximum concentration of 200 mg/mL in the s
olubility test conducted at BioReliance.
- Clinical signs of toxicity in test animals: Dose Level (mg/kg/day) Males Females
500 Piloerection, Diarrhea No signs
1000 Piloerection, Nasal charge, Lethargy, Hunched Piloerection, Nasal charge, Lethargy,
Hunched, Ataxia, Diarrhea
2000 Piloerection, Lethargy Piloerection, Lethargy
- Rationale for exposure: The route of exposure has been routinely used and is widely-accepted for use
in the mammalian bone marrow erythrocyte micronucleus assay.
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 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 induced a statistically significant
increase in the incidence of MnPCEs (Student's t-test, p- Ratio of PCE/NCE (for Micronucleus assay): No appreciable reductions in the PCEs/EC ratio were obs
erved in the test substance groups compared to the vehicle control group, indicating the test substance
did not induce cytotoxicity.
- Appropriateness of dose levels and route: see above for rationale for exposure
- Statistical evaluation: Group variances for 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.

Conclusions:

Under the conditions of the assay described in this report, 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether was concluded to be negative for the induction of micronucleated polychromatic erythrocytes.

Executive summary:

The test substance, 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether, was evaluated for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in rat bone marrow. Corn Oil was selected as the vehicle. Test and/or control substance formulations were administered once per day on two consecutive days at a dose volume of 10 mL/kg by oral gavage.

In the dose range-finding assay (DRF), the maximum dose tested was 2000 mg/kg/day. The dose levels tested were 500, 1000, and 2000 mg/kg/day in 3 of animals/sex. Based upon the results, the high dose for the definitive assay was 500 mg/kg/day, which was estimated to be the maximum tolerated dose (MTD)

The definitive assay dose levels tested were 125, 250, and 500 mg/kg/day.

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 administration of 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether at doses up to and including a dose of 125, 250, and 500 mg/kg/day was concluded to be negative in the Micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)