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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
The test item induces reverse mutation in bacteria, in the presence of S9 metabolism, under the reported experimental conditions. In the comet assay, it can be concluded that the test is negative for the induction of DNA damage in liver and duodenum, however; it is positive for the induction of DNA damage in stomach. The in vitro bacterial reverse mutaion assay and the in vivo mammalian alkaline comet assay are both positive.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation date: 04 January 2016 and Experimental Completion Date: 04 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 489, updated and adopted 26 September 2014.
Deviations:
yes
Remarks:
See below.
Principles of method if other than guideline:
The following deviations from the protocol occurred during the conduct of this study:
Event No. 233875: Animals in the Dose Range Finding assay were euthanized earlier than 3 to 4 Hours following second (and final) dose administration. All observations were performed for 1 and 2 hour following dose administration for toxicity determination. However, there was no requirement to record toxicity determination 3 to 4 hours following the final dose administration. Only the time recorded for the euthanasia was required.
Study Director Impact Statement: This deviation had no impact on the study outcome, since animals had no clinical signs of toxicity. Thus keeping them for one more hour would not have made a difference in toxicity or on the dose selection for the definitive assay.
GLP compliance:
yes
Type of assay:
mammalian comet assay
Species:
mouse
Strain:
other: Hsd:ICR (CD-1)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System
- Hsd:ICR (CD-1) mice were received from ENVIGO RMS, Inc., Frederick, MD on 27 January 2016 and 17 February 2016 (definitive assay).

- Age at initiation (weeks) for the dose range finding and the definitive test: 6 weeks for males and females
- Days of acclimation for the dose range finding and the definitive test: 5 days
- Weight at randomization (males for the DRF): 26.2 - 35.2 grams
- Weight at randomization (females for the DRF): 23.8 - 29.7 grams
- Weight at randomization (males for the Definitive test): 26.2 - 34.8 grams

Animal Receipt and Acclimation
Virus antibody-free (VAF) animals were acclimated as noted above and were judged to be healthy prior to utilization in the study.

Housing
Animals were housed in a controlled environment at 72 ± 3°F and 50 ± 20% relative humidity with a 12-hour light/dark cycle. The light cycle was not interrupted for study related activities. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air per hour. Animals of the same sex were housed up to three per Micro-Barrier cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system

Bedding, Food and Water
Heat treated hardwood chips (P.J. Murphy Forest Products) were used for bedding to absorb liquids. A certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients. Animals had free access to tap water, which met U.S. EPA drinking water standards [Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens. The results of bedding, food and water analyses are on file at BioReliance. There were no contaminants in the bedding, feed and water that were expected to interfere with the study.

Randomization and Animal Identification
Animals were assigned to groups using a randomization procedure. At the time of randomization, the weight variation per sex of all animals assigned to the study did not exceed ±20% of the mean weight. A randomization function within Excel™ was used to achieve random placement of animals throughout all groups. Following randomization, animals were identified by sequentially numbered ear tags. The cage card contained, at least, the animal number(s), sex, study number, treatment group number, dose level, test substance ID and route of administration. Cage cards were color coded by treatment group. Raw data records and specimens were also identified by the unique animal number.


Justification for the Test System
This species has been routinely used as an animal model of choice for the mammalian alkaline Comet assay. This strain was an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to the test substance.
Route of administration:
oral: gavage
Vehicle:
The vehicle used to deliver the test sustance to the test system was PEG 400.
- CAS number: 25322-68-3
- Supplier: Sigma Aldrich
- Lot/Batch number:BCBF6930V
- Expiration/Retest date: 28 Feb 2018
Details on exposure:
Preparation of Test Substance Dose Formulations
The test substance dose formulations were prepared at least once on each day of use. Each concentration was prepared by calibrating a suitable size amber vial with a PTFE stir bar to appropriate volume. An appropriate amount of the test substance was weighed and transferred into the vial and then QS to the final volume with the vehicle. The solution was mixed magnetically until homogenous. The formulation was vortexed with a vortex mixer and sonicated with a sonic bath for an appropriate amount of time until homogenous. The final dose formulation was stored at room temperature.


Dose Administration
All vehicle and test article dose formulations were administered at a volume of 20 mL/kg/day and positive control dose formulations were administered at a volume of 10 mL/kg by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The route has been routinely used and is widely-accepted for use in the mammalian Comet assay.
Duration of treatment / exposure:
2 consecutive days.
Twice daily approximately 2 hours apart on two consecutive days. Second day first dose administration occurred approximately 21 hours after the day one first administration
Frequency of treatment:
- For animals treated with the test item and / ot vehicle control: 2 times per day with approximately 2 hours apart.
- For animals treated with the positive control: Once approximately 3 to 4 hours prior to organ colection on Day 2.
Post exposure period:
not applicable
Remarks:
Doses / Concentrations:
0/250/500 and 1000 mg/kg/dose
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0/500/1000 and 2000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
6 males mice per dose per dose for the test group.
3 males mice for the positive control.
Control animals:
yes, concurrent vehicle
Positive control(s):
Methyl methanesulfonate (MMS) in 0.9% saline for injection was used as positive control.
The neat MMS was prepared in 0.9% saline for injection. The dosing formulation was prepared at a concentration of 40 mg/ml, just prior to use.

Methymethanesufonate (MMS) details):
- CAS number: 66-27-3
- Supplier: Sogma-Aldrich
- Lot/Batch number: MKBR6050V
- Expiration date: 31 March 2018

0.9% saline for injection details:
- CAS number: 7647-14-5
- Supplier: B. Braun Medical, Inc.
- Lot/Batch number: J4P213
- Expiration date: 31 May 2017
Tissues and cell types examined:
Liver, stomach and duodenum.
Details of tissue and slide preparation:
Tissue Collection for Comet Assay
All animals were euthanized 3 to 4 hours after the last dose (Study Day 2) by CO2 asphyxiation, and then, the following was performed:
• Animals were dissected and the liver, stomach and duodenum was extracted (removed) and collected.
• A section of the liver, stomach and duodenum was cut and placed in formalin for possible histopathology analysis.
• Another section of the liver, stomach and duodenum was placed in chilled mincing solution (Hanks’ balanced salt solution with EDTA and DMSO) and was used in preparation of cell suspensions and Comet slides.

Preparation of Cell Suspensions and Comet Slides
A portion of each dissected liver was placed in 3 mL of cold mincing buffer, then the liver was finely cut (minced) with a pair of fine scissors to release the cells. A portion of each dissected glandular stomach and duodenum was placed in 1 mL of cold mincing buffer then stomach and duodenum were scraped using a plastic spatula to remove the mucosa and rinsed with working mincing solution. The organs were then placed in cold mincing buffer and gently scraped to release the cells. Each cell suspension was strained through a Cell Strainer into a pre-labeled 50 mL polypropylene conical tube and the resulting liver, stomach and duodenum cell suspensions were placed on wet-ice. An aliquot of each of the suspensions was used to prepare the comet slides.

Preparation of Slides
From each liver, stomach and duodenal suspension, an aliquot of 2.5, 7.5 and 7.5 µL, respectively was mixed with 75 µL (0.5%) of low melting agarose (0.5%). The cell/agrose suspension was applied to microscope slides commercially available pre-treated multi-well slides. The slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify. At least two Trevigen, Inc 3-well slides were prepared per animal per tissue. Two slides/wells were used in scoring and the other wells were designated as a backup. Following solidification of agarose, the slides were placed in jars containing lysis solution.

Lysis
Following solidification of agarose, the slides were submerged in a cold lysis solution composed of 100 mM EDTA (disodium), 2.5 M sodium chloride, 10 mM tris hydroxymethyl aminomethane in purified water; pH 10; 1% Triton X-100 and 10% DMSO were added on the day of use. The slides were kept in this solution at least overnight at 2-8°C.

Unwinding
After cell lysis, slides/wells were washed with neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, pH ~7.5) and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with alkaline buffer composed of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water. The pH was > 13. All slides remained in the buffer for 20 minutes at 2-10C and protected from light, allowing DNA to unwind.

Electrophoresis
Using the same buffer, electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-10C and protected from light. The electrophoresis time was constant for all slides.

Neutralization
After completion of electrophoresis, the slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides (gels) were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for at least 4 hours and stored at room temperature with desiccant.

Staining
Slides were stained with a DNA stain (i.e., Sybr-gold) prior to scoring. The stain solution was prepared by diluting 1 L of Sybr-gold stain in 15 mL of 1xTBE (tris-boric acid EDTA buffer solution).

Evaluation criteria:
A test substance was considered to have induced a positive response if
- at least one of the group mean for the % tail DNA of the test substance doses exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
- when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01) and
- results of the group mean or of the individual animals of at least one group were outside the distribution of the historical negative control database for that tissue.
A test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met and there was direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue.
If the response was neither clearly positive nor clearly negative or in order to assist in establishing the biological relevance of a result, the data were evaluated by expert judgment and/or further investigations. Any additional work was only carried out following consultation with, and at the request of, the Sponsor.
In some cases, even after further investigations, the data set precluded making a conclusion of positive or negative, at which time the response was concluded to be equivocal. In such cases, the Study Dir. used sound scientific judgment and reported and described all considerations.
Biological significance of a positive, negative and equivocal result was based on the information on cytotoxicity at the target tissue. Where positive or equivocal findings were observed solely in the presence of clear evidence of cytotoxicity (e.g. histopathology evaluation, changes in clinical chemistry measures), the study was concluded as equivocal for genotoxicity unless there was enough information that was supportive of a definitive conclusion. In the case of a negative study outcome where there were signs of toxicity at all doses tested, further study at non-toxic doses may be advisable
Statistics:
Statistical Analysis
The median value of 150 counts of % Tail DNA, Tail moment and Tail migration were determined and presented for each animal in each treatment group for each organ. The mean and standard deviation of the median values only for % Tail DNA were presented for each treatment group. Statistical analysis was performed only for % Tail DNA.
In order to quantify the test substance effects on DNA damage, the following statistical analysis was performed:
• The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for % tail DNA generated for the vehicle and test substance groups were compared using Levene’s test (significant level of p  0.05). If the differences and variations between groups were found not to be significant, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p < 0.05). If Levene’s test indicated heterogeneous group variances (p  0.05), the suitability of a transformation of the original data was evaluated (e.g. using logarithm transformed values of the original data) in an attempt to meet the normality criteria. Afterwards, statistical analysis was performed using the parametric tests described above. If parametric tests were not acceptable, non-parametric statistical methods, (Kruskal Wallis and/or Mann Whitney test) may have been used in evaluation of data.
• A linear regression analysis was conducted to assess dose responsiveness in the test substance treated groups (p ≤ 0.01).
• A pair-wise comparison (Student’s T-test, p ≤ 0.05) was used to compare the positive control group to the concurrent vehicle control group.
Sex:
male/female
Genotoxicity:
positive
Remarks:
Negative for the induction of DNA damage in liver and duodenum, however; positive for the induction of DNA damage in stomach.
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
No mortality occurred at any dose level during the course of the dose range finding assay. No appreciable reductions in mean group body weights were seen in the test substance treated groups during the course of the study.

The following clinical singns were observed:
- Piloerection was observed in all males at all dose level (500/1000 and 2000 mg/kg/day; Treatment BID (two doses per day) to achieve a total (mg/kg/day)).
- Females were normal at all dose level (500/1000 and 2000 mg/kg/day; Treatment BID (two doses per day) to achieve a total (mg/kg/day)).

No mortality or significant differences in clinical observations were seen between the sexes, therefore the maximum tolerated dose for the definitive comet assay was set at 2000 mg/kg for males only.



RESULTS OF DEFINITIVE STUDY
No mortality occurred at any dose level during the course of the definitive assay. No appreciable reductions in mean group body weights were seen in the test substance treated groups during the course of the study.

The following clinical signs were observed:
- Males were normal after vehicle (PEG 400 administartion; Treatment BID (two doses per day) to achieve a total (mg/kg/day))
- Males treated at 500 mg/kg/day: Bloated Intestines were oebserved (a)
- Males treated at 1000 mg/kg/day: Bloated Intestines were oebserved (a)
- Males treated at 2000 mg/kg/day: Bloated Intestines were oebserved and diarrhea (a)
- Males treated at 40 mg/kg with the positive control (MMS) were normal
(a) Observation made at sacrifice

Comet Assay

Median values for the % Tail DNA,Tail moment and Tail migration (µm) forliver cellsare calculated per 150 cells for each animal.

The scoring results and a statistical analysis of data indicated the following:

-  The presence of ‘clouds’ in the test substance groups was ≤ 4.0% which was higher than the % of clouds in the vehicle control group (0.8%).

- Group variances for mean of medians of the % Tail DNA 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 and Dunnett’s post-hoc test, were used in the statistical analysis of data.

- No statistically significant response in the % Tail DNA (DNA Damage) in the test substance groups was observed relative to the concuruent vehicle control group (ANOVA, p > 0.005, Dunnett's post hoc analysis).

- No dose-dependent increase in the % Tai DNA was observed across three test substance doses (regression analysis, p > 0.01).

- The positive control, MMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the vehicle control groups (Student’s t‑test, p ≤ 0.05). 

- In the vehicle control group, % Tail DNA was within the historical vehicle control range for the liver. These results indicate that all criteria for a valid test, as specified in the protocol, were met. The scoring results and statistical analysis of data indicated the following: - The presence of "clouds" in the test substance groups was <= 43.7% which was higher than the % of clouds in the vehicle control group (31.3%). - Group variance for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene's test. The test indicate that there was no significant difference in the group variance (p > 0.05) therefore the parametric approach, ANOVA and Dunnett's post-hoc test, were used in the statistical analysis of data. - Statistically significant response in the % Tail DNA (DNA Damage) in the test substance groups (1000 and 2000 mg/kg/day) was observed relative to the concurrent vehicle control group (ANOVA, p < 0.05, Dunnett's post hoc analysis). - Dose dependant increase in the % Tail DNA ( DNA Damage) was observed across three test substance doses (regression analysis, p <0.01). - The positive control, MMS, induced a statistically significant increase in the % Tail DNA in stomach cells as compared to the vehicle control groups (Student's t-test, p < = 0.05). - In the vehicle control group, % Tail was within the historical vehicle control range for the stomach, These results indicate that all criteria for a valid test, as specified in the protocol were met. Median values for the % Tail DNA,Tail moment and Tail migration (µm) forduodenal cellsare calculated per 150 cells for each animal. The scoring results and a statistical analysis of data indicated the following:

-  The presence of ‘clouds’ in the test substance groups was ≤ 16.5% which was higher than the % of clouds in the vehicle control group (10.3%).

- Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene's test. The test indicated that there was significant difference in the group variance (p < 0.05) therefore the suitability of a transformation of the original data was evaluated (e.g. using logarithm transformed values of the original data) in an attempt to meet the normality.

Afterwards, the parametric approach, ANOVA and Dunnett's post-hoc test, was used in the statistical analyis of data.

- No statistically significant response in the % Tail DNA (DNA Damage) in the test substance groups was observed relative to the concurrent vehicle control group (ANOVA, p < 0.05, Dunnett's post hoc analysis).

- No dose -dependant increase in the % Tail DNA was observed across three test substances doses (regression analysis, p >0.01).

- The positive control MMS, induced a statistically significant incease in the % Tail DNA in duodenal cells as compared to the vehicle control groups (Student's t-test, p <= 0.05).

- In the vehicle control group, % Tail DNA was within the historical vehicle control range for the duodenum. These results indicate that all criteria for a valid test, as specified in the protocol, were met.

Dosing Formulation Analysis

The results of the analysis indicate that the actual mean concentrations of the analyzed formulation samples, 25 mg/ml, 50 mg/ml and 100 mg/ml, were 104.1, 101.6 and 97.7%, respectively, with a S/L ratio of > 0.925. This indicates that the formulations were accurately prepared. No Test Substance was detected in the vehicle control sample. Additionally, 2,5-bis[(glycidyloxy)phenyl]octahydro-4,7-methano-5h-indene in PEG 400, at concentrations of 26.0 and 97.7 mg/mL, was stable at room temperature for at least 2.53 hours. In addition, 2,5-bis[(glycidyloxy)phenyl]octahydro-4,7-methano-5h-indene in PEG 400, at concentrations of 24.1 and 95.1 mg/mL, was found to be stable at room temperature for at least 5.08 hours.

Histopathology Analysis

Stomach tissues from male mice were prepared from the test article, positive control and vehicle control treated animals because of a positive finding. The samples were sent to the Test Site for histopathology analysis. The results of the analysis found that there were no test article-related microscopic findings in the stomachs of male CD-1 mice.

Conclusion

Under the conditions of the assay described in this report, the test sustance concluded to be negative for the induction of DNA damage in liver and duodenum, however; positive for the induction of DNA damage in stomach.

Conclusions:
Interpretation of results (migrated information): positive
Under the conditions of the assay described in this report, 2,5-bis[(glycidyloxy)phenyl]octahydro-4,7-methano-5h-indene concluded to be negative for the induction of DNA damage in liver and duodenum, however; positive for the induction of DNA damage in stomach.

Executive summary:

The test substance, was evaluated for its genotoxic potential in the Comet assay to induce DNA damage in liver, stomach and duodenum cells of male mice. PEG 400 was selected as the vehicle. Test and/or vehicle control article formulations were administered at a dose volume of 10 mL/kg with BID dosing approximately 2 hours apart and positive control formulations were

administered 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. The additional dose levels tested were 1000 and 500 mg/kg/day in 3 mice/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg, which was estimated to be the maximum tolerated dose (MTD).

The definitive assay dose levels tested were 500, 1000, and 2000 mg/kg/day.

The test substance gave a negative (non-DNA damaging) response in this assay in liver and duodenum for the males in % Tail DNA. None of the test substance treated animal slides had significant increases in the % Tail DNA compared to the respective vehicle controls. The vehicle control % Tail DNA was within Testing Facility’s historical range and positive control had a statistically significant increase in % Tail DNA compared to the vehicle control. The test substance gave a positive (DNA-damaging) response in this assay in stomach for the males in % Tail DNA. The mid and high dose test substance treated animal slides had significant increases in the % Tail DNA compared to the respective vehicle controls. The vehicle control % Tail DNA was within Testing Facility’s historical range and positive control had a statistically significant increase in % Tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver, duodenum and stomach.

Under the conditions of this study, the administration of the test item at doses up to and including a dose of 2000 mg/kg/day did not cause a significant increase in DNA damage in liver and duodenum, however; it did cause a significant increase in DNA damage in stomach relative to the concurrent vehicle control.

Therefore, the test substance was concluded to be positive in the in vivo Comet Assay

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vivo:

A bacterial reverse mutation test performed according to OECD guidelines and GLP principles is available. TK 30250 was found to be mutagenic in the bacterial reverse mutation in presence of S9 metabolic activation.

The in vivo mammalian alkaline comet assay turn out to be also positive for the induction of DNA Damage in stomach.


Justification for selection of genetic toxicity endpoint
A reliable in vitro bacterial reverse mutation test was performed according to OECD/EC guidelines 471 followed by a in vivo mammalian alkaline comet assay in mice (OECD 489).

Justification for classification or non-classification

Based on the above mentioned results the substance does need to be classified as mutagenic, in vitro (OECD 471) and in vivo (OECD 489), according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).

TACTIX 556 is classified as mutagenic category 2.