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Administrative data

Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-[3-(aminomethyl)phenyl]methanamine; 2,4-bis[({[3-(aminomethyl)phenyl]methyl}amino)methyl]phenol; 2,6-bis[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-[(4-hydroxyphenyl)methyl]phenol; 2-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-[(4-hydroxyphenyl)methyl]phenol; 2-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]phenol; phenol
EC Number:
701-207-5
Cas Number:
1950616-36-0
Molecular formula:
(C8 H12 N2 . C6 H6 O . C H2 O)x
IUPAC Name:
1-[3-(aminomethyl)phenyl]methanamine; 2,4-bis[({[3-(aminomethyl)phenyl]methyl}amino)methyl]phenol; 2,6-bis[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-[(4-hydroxyphenyl)methyl]phenol; 2-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-[(4-hydroxyphenyl)methyl]phenol; 2-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]phenol; phenol
Specific details on test material used for the study:
Identity: EPIKURE 105
Alternative names: EPIKURE
Chemical name: Methanal reaction products with 1,3-bis(aminomethyl)benzene and hydroxybenze
Batch number: DG6L50992A
CAS number: 1950616-36-0
Appearance: Brown-yellow liquid
Storage conditions: Room temperature (15-25oC)
Purity/Assay: 100% UVCB
Expiry date: 27 January 2022
Correction factor for formulation: None, used as supplied
Test item received: 30 January 2020

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
The animals were obtained from Charles River UK Limited, Margate, Kent, England.
On the first day of dosing, animals used on the study weighed between:
Preliminary toxicity test: Males weighed between 230 g - 294 g.
Females weighed between 182 g - 228 g.
Comet test: Males weighed between 218 g - 251 g.
Animal age on dispatch and on Day 1 of dosing was:
Preliminary toxicity test: On Dispatch Males and females ca 42 - 48 days old.
Day 1 Males and females ca 49 - 63 days old.
Comet test: On Dispatch Males ca 42 - 48 days old.
Day 1 Males ca 48 - 54 days old.
Sex:
male/female
Details on test animals or test system and environmental conditions:
After arrival the weight of the animals was checked and found to be acceptable. The animals
were randomly assigned to groups and given a unique tail mark. Each group was kept with
the sexes separated in cages. The animals were maintained in a controlled environment with
the thermostat and relative humidity target ranges set at 20 to 24°C and 40 to 70%
respectively. Temperature and humidity were within range throughout the study. The room
was illuminated by artificial light for 12 hours per day.
All animals were allowed free access to pelleted Envigo Teklad 2014C diet and tap water ad
libitum.
Food, chew blocks and tap water are routinely analysed for quality at source. All animals
were given access to small soft white untreated wood (aspen) chew blocks and a plastic
shelter for environmental enrichment and were acclimatised for a minimum of 5 days.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
The test item was administered on two occasions approximately 24 hours apart.
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate

Examinations

Tissues and cell types examined:
Liver, duodenum, glandular stomach.
Details of tissue and slide preparation:
Glass slides were dipped in 1% normal melting point agarose and left to air dry prior to addition of the cell suspension layer.
For each tissue type, an appropriate dilution of the cell suspensions were made and mixed with the appropriate volume of 0.5% low melting point agarose. A 75µL aliquot of the cell/agar mix was dispensed onto the appropriate pre-dipped slides and cover-slipped. Cell concentrations used documented below:
Tissue Cell suspension volume (µL) Low melting point agarose (LMPA)
volume (µL)
Liver 75 925
Duodenum 100 900
Glandular stomach 100 900
Once the agar had set the cover slips were removed and the slides immersed in chilled lysis
solution in a light proof box. These were stored at 2 - 8ºC overnight prior to electrophoresis.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly
positive if: a) at least one of the test doses exhibits a statistically significant increase
compared with the concurrent vehicle control, b) the increase is dose-related when evaluated
with an appropriate trend test, and c) any of the results are outside the distribution of the
historical vehicle control data. When all of these criteria are met, the test item is then
considered able to induce DNA strand breakage in the tissues studied in this test system.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative
if: a) none of the test concentrations exhibits a statistically significant increase compared with
the concurrent vehicle control, b) there is no concentration-related increase when evaluated
with an appropriate trend test and c) all results are inside the distribution of the historical
vehicle control data. The test item is then considered unable to induce DNA strand breakage
in the tissues studied in this test system.
There is no requirement for verification of a clearly positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above and in
order to assist in establishing the biological relevance of a result, the data should be evaluated
by expert judgement and/or further investigations. Scoring additional cells (where
appropriate) or performing a repeat experiment possibly using modified experimental
conditions (e.g. dose spacing, other routes of administration, other sampling times or other
tissues) could be useful.
In rare cases, even after further investigations, the data set will preclude making a conclusion
of positive or negative results, and will therefore be concluded as equivocal.
To assess the biological relevance of a positive or equivocal result, cytotoxicity at the target
tissue should also be discussed. Histopathological informa

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Evidence of DNA strand breaks in the duodenum and glandular stomach at 2000 mg/kg/day only when dose-related presence of hedgehog cells and stomach and duodenum with necrosis and apoptosis.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
It is concluded that EPIKURE 105 has not shown any evidence of causing an increase in
DNA strand breaks in the liver of male Crl: CD(SD) rats when administered orally by gavage
in this in vivo test procedure.
It is concluded that EPIKURE 105 has shown evidence of causing an increase in DNA strand
breaks in the duodenum and glandular stomach of Crl:CD(SD) rats at 2000 mg/kg/day only
when administered orally by gavage in this in vivo test procedure. However, EPIKURE 105
is considered equivocal, due to the dose-related presence of hedgehog cells and the
histopathological examination of stomach and duodenum showing evidence of necrosis and
apoptosis respectively.
Executive summary:

Introduction


This study was designed to assess the potential of EPIKURE 105 to induce DNA strand breaks in the liver, duodenum and glandular stomach of Crl: CD(SD) rats.
All animals in the vehicle control and test item dose groups were dosed orally by gavage using a dose volume of 10 mL/kg on two occasions, the second dose being administered approximately 24 hours after the first dose and 3 hours before sampling.
The positive control group were dosed orally by gavage using a dose volume of 10 mL/kg on a single occasion approximately 3 hours before sampling.
On the basis of the results from the preliminary toxicity test, dose levels of 500, 1000 and 2000 mg/kg/day were selected for the comet test. No substantial differences in toxicity between sexes were observed in the preliminary toxicity test; therefore, in line with current guidelines the main test was performed using male animals only.
The vehicle control group received propylene glycol and the positive control group received Ethyl Methanesulphonate at 200 mg/kg.
Cell suspensions from the tissues were obtained from animals in the vehicle control group and in each of the test item groups 3 hours after administration of the second dose. Cell suspensions from animals in the positive control group were obtained approximately 3 hours after a single dose.
Following electrophoresis three slides per animal per tissue were analysed for comets.
Slides were visualised by staining with SYBR GOLD® via fluorescence microscopy.
150 morphologically normal cells were analysed for the presence of comets per animal per tissue.
DNA strand breaks were assessed by comparing the group % tail intensities (% TI) from test item treated animals with the concurrent vehicle control values. The slides were also examined for any overt toxicity, e.g. an increase in background debris and/or an increase in the incidence of excessively damaged cells (i.e. Hedgehog cells). These cells were excluded from the analysis, along with any cells that had unusual staining artefacts.
Sections of the duodenum and glandular stomach from the vehicle control animals and animals administered EPIKURE 105 at 2000 mg/kg/day were processed for histopathological examination and assessed for signs of cytotoxicity, necrosis and apoptosis.



Results


The mean concentrations of EPIKURE 105 for all main test formulations were within -14% of the nominal concentration, confirming the accuracy of formulation. The difference from mean for individual values remained within 5%, confirming precise analysis, with the exception of group 2 which had a single % difference from mean value outside of limits (5.42 %). As the deviation was minor and as it had been confirmed that the standard test limit had been achieved, satisfying the test guidelines the formulations were considered acceptable for dosing.
The vehicle control group % tail intensity (%TI) values for the liver, duodenum and glandular stomach of male rats were within or close to the current vehicle historical control range for the individual tissues (95% confidence limits). The positive control compound, EMS, produced a statistically significant increase (p≤0.001) in the median %TI when compared to vehicle control values in all tissues analysed.
There were no statistically significant increases, or trend, observed in the %TI in the liver of male Crl: CD(SD) rats administered EPIKURE 105 at 500, 1000 or 2000 mg/kg/day. The group median %TI values were all within the current vehicle historical control range (95% confidence limits).
There was no statistically significant increase observed in the median %TI in the duodenum or glandular stomach of male Crl: CD(SD) rats administered EPIKURE 105 at 500 or 1000 mg/kg/day. The group median %TI values were all within the current vehicle historical control range (95% confidence limits).
However, statistically significant increases were observed in the %TI in the duodenum and glandular stomach of male Crl: CD(SD) rats administered EPIKURE 105 at 2000 mg/kg/day (p≤0.001). In the glandular stomach, the group median %TI values were marginally within the current vehicle historical control range (95% confidence limits), with 2/6 individual values outside of the historical control range (95% confidence limits) for individual animals.
In the duodenum, the group median %TI values, and all individual animal values were outside the current vehicle historical control range (95% confidence limits). There was a statistically significant trend observed from groups 1 to 4 (p≤0.001) in both tissues.
There were no hedgehog cells observed in the liver of any animal.
There was a substantial increase in the number of hedgehog cells observed in the duodenum of male Crl: CD(SD) rats administered EPIKURE 105 at 2000  mg/kg/day (group mean: vehicle, 500, 1000 and 2000 mg/kg/day: 0.0%, 0.0%, 0.0% and 22.41%, respectively).
There was a substantial increase in the number of hedgehog cells observed in the glandular stomach of male rats administered EPIKURE 105 at 2000 mg/kg/day (group mean vehicle, 500, 1000 and 2000 mg/kg/day: 0.0%, 0.0%, 0.0% and 10.18% respectively).
Histopathological examination showed necrosis of the glandular epithelium of the stomach, close to the limiting ridge, in one male animal receiving EPIKURE 105 at 2000 mg/kg/day.
This finding was accompanied by slight infiltration of mixed inflammatory cells. The duodenum showed apoptosis within the cells located within the lamina propria in all animals receiving EPIKURE 105 at 2000 mg/kg/day.


Discussion


There was clearly no effect on the liver at any dose level or the duodenum and glandular stomach of animals administered EPIKURE 105 at 500 and 1000 mg/kg/day.
Hedgehogs cells are heavily damaged cells which exhibit a specific morphology (a small or non-existent head and a large diffuse tail); they are considered to be associated with cytotoxicity, necrosis or apoptosis; however, the exact aetiology of hedgehog cells is ambiguous. Other evidence suggests they may be caused by mechanical/enzyme induced damage during sample preparation or extreme effects of test chemical genotoxicity (OECD 489, 2016). There were no hedgehog cells observed in the vehicle control animals; as all tissue samples were prepared using identical methods, the cause of the hedgehog cells is not considered to be mechanical/enzyme induced damage which occurred during sample preparation.
The hedgehog cells observed in the duodenum and glandular stomach of male animals administered EPIKURE 105 at 2000 mg/kg/day are considered to be directly correlated to the increases in the %TI. Although the precise aetiology of hedgehog cells is not well understood, it is very clear that they, or their cause, have in this study confounded the comet response at 2000 mg/kg/day.
There was slight evidence of apoptosis and necrosis related to treatment observed following the histopathological examination of the duodenum and stomach of male Crl: CD(SD) rats administered EPIKURE 105 at 2000 mg/kg/day.
The OECD 489 (2016) guidance is clear that in the presence of clear cytotoxicity any relevant increases should be interpreted with care. It is considered therefore, that the increases obtained in the duodenum and glandular stomach are unlikely to be of genotoxic origin.


Conclusion


It is concluded that EPIKURE 105 has not shown any evidence of causing an increase in DNA strand breaks in the liver of male Crl: CD(SD) rats when administered orally by gavage in this in vivo test procedure.
It is concluded that EPIKURE 105 has shown evidence of causing an increase in DNA strand breaks in the duodenum and glandular stomach of Crl:CD(SD) rats at 2000 mg/kg/day only when administered orally by gavage in this in vivo test procedure. However, EPIKURE 105 is considered equivocal, due to the dose-related presence of hedgehog cells and the histopathological examination of stomach and duodenum showing evidence of necrosis and apoptosis respectively.