Registration Dossier

Administrative data

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)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)
Type of assay:
other: Mammalian Erythrocyte Micronucleus Assay in Rats

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid: viscous
Details on test material:
Identification:
1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether
Appearance/Physical state:
clear, colorless, viscous liquid
Batch:
WA 1508
Purity:
100% UVCB
Expiry date:
01 January 2021
Storage conditions:
room temperature, in the dark
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.

Test animals

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 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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.