(E)-1,2-difluoroethylene

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 17th September 2020, experimental completion date 12th November 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 17 September 2020 ,experimental completion date 12 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (2011) EMA/CHMP/ICH/126642/2008. Guideline S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Commission Regulation No. 2017/735. Method B.62: Mutagenicity – In Vivo Mammalian Alkaline Comet Assay. OJ L 112/180.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Identity: TKN1
Batch number: N1200225
Appearance: Colourless gas
Storage conditions: At ambient temperature (15 to 25°C)
Purity: 100%
Re-assay date: 20th September 2022

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD) rat

Sex:

male

Details on test animals or test system and environmental conditions:

All animals used on this study were Crl:CD (SD) rats. On the first day of dosing, animals (males) used on the study weighed between 243.2 g and 296.2 g. Animal age on dispatch was over a range of approximately 42-48 days old, on day 1 of testing ages ranged from approximately 55-61 days old.

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. The animals were kept in a controlled environment with the thermostat and relative humidity within target ranges of 20 to 24°C and 40 to 70% respectively. 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.

All animals were given access to small soft white untreated wood (Aspen) chew blocks and a red plastic shelter for environmental enrichment and were acclimatised for a minimum of 5 days. Food, chew blocks and tap water are routinely analysed for quality at source.

Animals were inspected at least twice daily for evidence of ill-health, mortality or reaction to treatment. Any clinical signs were recorded at the time in respect of nature and severity, date and time of onset.

Animal weights were recorded for each animal from Day -5 and on the day of necropsy.

Route of administration:

inhalation: gas

Vehicle:

sterile air

Details on exposure:

Before commencement of treatment the system was characterized at the target exposure
aerosol concentrations without animals in order to demonstrate reproducibility of atmosphere
concentration.

The test item (gas) was metered to the exposure chamber directly from a pressurized cylinder.

The flow-through exposure chamber was modular in construction, made from an aluminium
alloy. It comprised of a base unit, an animal exposure section with 20 exposure ports, and a
top section incorporating a central aerosol inlet with a tangential air inlet.

During exposure, the animals were held in restraining tubes with their snouts protruding from
the ends of the tubes into the exposure chambers; any unused exposure ports were closed
with blanking plugs. Animals were acclimated to the restraints for a period of 3 days prior to
use in the system.

The temperature in the exposure chamber was monitored continuously during exposure and
recorded at 30 minute intervals.

At least 3 samples were withdrawn from each test chamber via the animal exposure ports of
the inhalation systems on each day of exposure, in order to estimate the concentration of the
test item in the chamber air.

Samples were analysed using a GC method validated with respect to the specificity of analysis,
limit of detection, linearity of detector response and repeatability.

Duration of treatment / exposure:

The test item was administered on three occasions (6-hour exposure), the second dose being administered approximately 24 hours after the first dose, with the third dose being administered approximately 21 hours after the second dose. Sampling was performed approximately 3 hours after the completion of the 6-hour exposure period on Day 3.

Frequency of treatment:

3 test item administrations (3 days)

Post exposure period:

Following dosing, the animals were examined regularly during the working day, any mortalities or clinical signs of reaction during the experiment were recorded.
Animals from the vehicle control and test item groups were killed approximately 3 hours after completion of the third dose. In addition, animals in the positive control group (comet phase) were killed 3 hours after a single dose. All animals were killed by an overdose of anaesthetic.

Dose / conc.:

0 ppm

Remarks:

Vehicle (sterile air) (group 1)

Dose / conc.:

30 000 ppm

Remarks:

Group 2

Dose / conc.:

60 000 ppm

Remarks:

Group 3

Dose / conc.:

120 000 ppm

Remarks:

Group 4

Dose / conc.:

200 other: mg/kg

Remarks:

Dose of positive control substance, EMS

No. of animals per sex per dose:

Group 1 (vehicle control) and groups 2, 3, and 4 each consisted of 6 male rats. Group 5 (positive control group) consisted of 3 male rats.

Control animals:

yes, concurrent vehicle

Positive control(s):

EMS - Ethyl methanesulfonate administered to group 5 (positive control group) orally at 200 mg/kg on one occasion 3 hours prior to termination.

Tissues and cell types examined:

Cell suspension from sections of the liver, kidney, urinary bladder and lungs

Details of tissue and slide preparation:

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.

Sections of the liver, kidney, urinary bladder and lungs were placed into ice cold mincing solution, and all samples were stored on ice before processing for Comet analysis. Single cell suspensions were prepared using a tissue specific method.

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.

Comet slides were prepared from all cell suspensions.

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.

Sections of the liver, kidney, urinary bladder and lungs were stored in 10% buffered formalin

Electrophoresis:

The slides were placed onto a dry, level platform of a horizontal electrophoresis unit containing chilled electrophoresis buffer. The slides from each tissue from each treatment group were spread across the platform to avoid any positional effects.

The buffer reservoir was filled with electrophoresis buffer until the slides were covered and the nucleoids were left to unwind for 20 minutes at approximately 2 - 10˚C.

After alkali unwinding the slides were electrophoresed at 18V, approximately 300mA (between 0.7 and 1.0 V/cm) for 30 minutes.

Once electrophoresis was complete the slides were washed with neutralising buffer and stored, refrigerated in lightproof boxes with moistened tissue paper to prevent agar dehydration.

Microscopic Examination:

Coded slides were examined by staining with SYBR GOLD® and visualised under a fluorescence microscope. The comet images from the microscope were captured via a CCD camera and measured using Perceptive Instruments COMET IVTM image analysis system.

Initially the slides were 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.

Fifty cells were scored per slide to give a total number of 150 cells per tissue per animal. The extent of DNA migration and hence damage is reflected by:

% Tail intensity, defined as the fluorescence detected by image analysis in the tail, which is
proportional to the amount of DNA that has moved from the head region into the comet tail.

Evaluation criteria:

Interpretation of Results:
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.

To assess the biological relevance of a positive or equivocal result, cytotoxicity at the target tissue should also be discussed. Histopathological information can help in the interpretation of a positive result in the comet assay.

Statistics:

Tail intensity data was supplied for each slide. The median of the log-transformed tail intensities from each slide was averaged to give an animal summary statistic. If the median value on a slide is zero, a small constant (0.0001) was added before taking the logarithm and calculating the average for the animal. This animal average was used in the statistical analysis.

Data was analysed using one-way analysis of variance (ANOVA) with the fixed factor for treatment group. The positive control group was excluded from this analysis. Levene’s test for equality of variances across the groups was also performed. Where this shows evidence of heterogeneity (p≤0.01), the data was rank-transformed prior to analysis using ANOVA on the ranks. Comparisons between each treated group and control was made using Dunnett’s test. The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p-value is reported. In addition, a linear contrast was used to test for an increasing dose response.

The positive control group was compared to the control group using a two-sample t-test. Levene’s test for equality of variances between the groups was also performed. Where this shows evidence of heterogeneity (p≤0.01), the data was rank-transformed prior to analysis using a two-sample t-test on the ranks. The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p-value is reported.

.

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

TKN1 has shown evidence of causing an increase in DNA strand breaks in the bladder, lung, kidney and liver of male Crl:CD (SD) rats when administered via inhalation in this in vivo test procedure.

Toxicity:

yes

Remarks:

Statistically significant increase in Hedgehog cells in bladder, lung, kidney and liver

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Inhalation Analysis:
Mean achieved atmosphere concentrations were 104, 106 and 102% of target for groups 2, 3 and 4 respectively.

Main Test:
The test was carried out in male animals only. No mortalities were observed throughout the duration of the test.

Clinical Signs:
Animals were treated with TKN1 at dose levels of 30,000, 60,000 and 120,000 ppm.
Clinical signs including red staining on the head and wet fur were observed in the vehicle control animals.
At 30,000 and 120,000 ppm, clinical signs included red staining on the head and wet fur. At 60,000 ppm, clinical signs included red staining on the head, eyes and nose.
There were no incidences of group mean body weight loss (Day 1 to Day 3. Small incidences of bodyweight loss between individual weighings were observed in male animals in all groups.



Comet Phase:
% Tail Intensity:

The vehicle control group % tail intensity (%TI) values for the bladder, lung, kidney and liver of male rats were within the current vehicle historical control range for the individual tissue (95% confidence limits). The positive control compound, Ethyl methanesulfonate, produced statistically significant increases in the median % TI when compared to vehicle control values (p≤0.001).

Bladder:

There were no statistically significant increases in the median % TI were observed in the bladder of male Crl:CD (SD) rats administered TKN1 at 30,000 and 60,000 ppm, compared to vehicle control values. The group mean and all individual %TI values from rats administered TKN1 at 30,000 ppm were within the current vehicle historical control range (95% confidence limits). The group mean %TI value from rats administered TKN1 at 60,000 ppm was outside of the current vehicle historical control range (95% confidence limits), but this was largely attributed to two animals.

Statistically significant increases in the median % TI was observed in the bladder of male rats administered TKN1 at 120,000 ppm compared to vehicle control values (p≤0.001). The group mean and all individual %TI values were outside of the current vehicle historical control rage (95% confidence limits). There was also a statistically significant trend observed from groups 1 to 4 (p≤0.001).


Lung:

There were no statistically significant increases in the median % TI were observed in the lung of male Crl:CD (SD) rats administered TKN1 at 30,000 and 60,000 ppm, compared to vehicle control values. Additionally, the group means and all individual %TI values were within the current vehicle historical control range (95% confidence limits).

A statistically significant increase in the median % TI was observed in the lung of male rats administered TKN1 at 120,000 ppm compared to vehicle control values (p≤0.001). The group mean and all individual %TI values were outside of the current vehicle historical control range (95% confidence limits). There was also a statistically significant trend observed from groups 1 to 4 (p≤0.001).


Kidney:

There were no statistically significant increases in the median % TI were observed in the kidney of male Crl:CD (SD) rats administered TKN1 at 30,000 ppm, compared to vehicle control values. The group mean %TI value was outside of the current vehicle historical control range (95% confidence limits), but this was largely attributed to two animals.

Statistically significant increases in the median % TI was observed in the kidney of male rats administered TKN1 at 60,000 and 120,000 ppm compared to vehicle control values (p≤0.001). The group means and all individual %TI values were outside of the current vehicle historical control range (95% confidence limits). There was also a statistically significant trend observed from groups 1 to 4 (p≤0.001).


Liver:

No statistically significant increases in the median % TI were observed in the liver of male Crl:CD (SD) rats administered TKN1 at 30,000 and 60,000 ppm, compared to vehicle control values. The group means and all individual %TI values were within the current vehicle historical control range (95% confidence limits).

A statistically significant increase in the median % TI was observed in the liver of male rats administered TKN1 at 120,000 ppm compared to vehicle control values (p≤0.001). The group mean and all individual %TI values were outside of the current vehicle historical control range (95% confidence limits). There was also a statistically significant trend observed from groups 1 to 4 (p≤0.001).


Hedgehog Cell Data:

Bladder:
There was a substantial increase in the number of hedgehog cells observed in the bladder of male rats administered TKN1 at 60,000 and 120,000 ppm (group mean vehicle, 30,000, 60,000 and 120,000 ppm: 0.00%, 0.00%, 2.28% and 8.72% respectively).

Lung:

There was a substantial increase in the number of hedgehog cells observed in the lung of male rats administered TKN1 at 120,000 ppm (group mean vehicle, 30,000, 60,000 and 120,000 ppm: 0.00%, 0.00%, 0.00% and 7.98% respectively).

Kidney:
There was a substantial increase in the number of hedgehog cells observed in the kidney of male rats administered TKN1 at 60,000 and 120,000 ppm (group mean vehicle, 30,000, 60,000 and 120,000 ppm: 0.0%, 0.33%, 5.56% and 5.76% respectively).


Liver:

There was a substantial increase in the number of hedgehog cells observed in the liver of male rats administered TKN1 at 120,000 ppm (group mean vehicle, 30,000, 60,000 and 120,000 ppm: 0.0%, 0.0%, 0.55% and 6.83% respectively).

For full summary of the results of the comet phase and stastical analysis please refer to attachment in "Attached background material" section, results for individual animals are also included.

Conclusions:

It is concluded that TKN1 has shown evidence of causing an increase in DNA strand breaks
in the bladder, lung, kidney and liver of male Crl:CD (SD) rats when administered via
inhalation at above 30000 ppm in this in vivo test procedure.

Executive summary:

Summary

This study was designed to assess the potential of TKN1 to induce DNA strand breaks in the liver, kidney, urinary bladder and lungs and also assess the potential induction of micronuclei in the bone marrow cells of Crl:CD (SD) rats.

Animals were treated with TKN1 via the inhalation route on three occasions (6-hour exposure), the second dose being administered approximately 24 hours after the first dose, with the third dose being administered approximately 21 hours after the second dose. Sampling was performed approximately 3 hours after the completion of the 6-hour exposure period on Day 3.

Dose levels of 30,000, 60,000 and 120,000 ppm were selected for the test. The test was performed using male animals only.

Animals in the comet phase positive control group were dosed orally on a single occasion using a dose volume of 10 mL/kg.

The vehicle control group received sterile air and the positive control group for the comet phase received Ethyl Methanesulfonate at 200 mg/kg.

Comet Phase

Cell suspensions from each tissue were obtained from animals in the vehicle control group and in each of the test item groups 3 hours after the completion of the 6-hour exposure period on Day 3. Cell suspensions from animals in the positive control group were obtained approximately 3 hours after a single dose.

Sections of the liver, kidney, urinary bladder and lungs from the vehicle control animals and animals administered TKN1 at 30,000, 60,000 and 120,000 ppm were stored in 10% buffered formalin but not analysed at the request of the sponsor.

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 mean and median % tail intensities (% TI) from TKN1 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.

Conclusions

It is concluded that TKN1 has shown evidence of causing an increase in DNA strand breaks in the bladder, lung, kidney and liver of male Crl:CD (SD) rats when administered via inhalation in this in vivo test procedure.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

EC Commission Regulation No. 2017/735. Method B.12: Mutagenicity - In vivo
Mammalian Erythrocyte Micronucleus Test. OJ L 112/54.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH (2011) EMA/CHMP/ICH/126642/2008. Guideline S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

(E)-1,2-difluoroethylene

EC Number:

216-629-0

EC Name:

(E)-1,2-difluoroethylene

Cas Number:

1630-78-0

Molecular formula:

C2H2F2

IUPAC Name:

(E)-1,2-difluoroethene

Test material form:

gas

Specific details on test material used for the study:

Identity: TKN1
Batch number: N1200225
Appearance: Colourless gas
Storage conditions: At ambient temperature (15 to 25°C)
Purity: 100%
Re-assay date: 20th September 2022

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD) rat

Sex:

male

Details on test animals or test system and environmental conditions:

Group 1 (vehicle control) and groups 2, 3, and 4 each consisted of 6 male rats.

All animals used on this study were Crl:CD (SD) rats. On the first day of dosing, animals (males) used on the study weighed between 243.2 g and 296.2 g. Animal age on dispatch was over a range of approximately 42-48 days old, on day 1 of testing ages ranged from approximately 55-61 days old.

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. The animals were kept in a
controlled environment with the thermostat and relative humidity within target ranges of 20
to 24°C and 40 to 70% respectively. 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.

All animals were given access to small soft white untreated wood (Aspen) chew blocks and a
red plastic shelter for environmental enrichment and were acclimatised for a minimum of
5 days. Food, chew blocks and tap water are routinely analysed for quality at source.

Animals were inspected at least twice daily for evidence of ill-health, mortality or reaction to treatment. Any clinical signs were recorded at the time in respect of nature and severity, date and time of onset.

Animal weights were recorded for each animal from Day -5 and on the day of necropsy.

Administration / exposure

Route of administration:

inhalation: gas

Vehicle:

sterile air

Details on exposure:

Before commencement of treatment the system was characterized at the target exposure
aerosol concentrations without animals in order to demonstrate reproducibility of atmosphere
concentration.

The test item (gas) was metered to the exposure chamber directly from a pressurized cylinder.

The flow-through exposure chamber was modular in construction, made from an aluminium
alloy. It comprised of a base unit, an animal exposure section with 20 exposure ports, and a
top section incorporating a central aerosol inlet with a tangential air inlet.

During exposure, the animals were held in restraining tubes with their snouts protruding from
the ends of the tubes into the exposure chambers; any unused exposure ports were closed
with blanking plugs. Animals were acclimated to the restraints for a period of 3 days prior to
use in the system.

The temperature in the exposure chamber was monitored continuously during exposure and
recorded at 30 minute intervals.

At least 3 samples were withdrawn from each test chamber via the animal exposure ports of
the inhalation systems on each day of exposure, in order to estimate the concentration of the
test item in the chamber air.

Samples were analysed using a GC method validated with respect to the specificity of analysis,
limit of detection, linearity of detector response and repeatability.

Duration of treatment / exposure:

The test item was administered on three occasions (6-hour exposure), the second dose being
administered approximately 24 hours after the first dose, with the third dose being
administered approximately 21 hours after the second dose. Sampling was performed
approximately 3 hours after the completion of the 6-hour exposure period on Day 3.

Frequency of treatment:

3 test item administrations (3 days)

Post exposure period:

Following dosing, the animals were examined regularly during the working day, any mortalities or clinical signs of reaction during the experiment were recorded.
Animals from the vehicle control and test item groups were killed approximately 3 hours after completion of the third dose. In addition, animals in the positive control group (comet phase) were killed 3 hours after a single dose. All animals were killed by an overdose of anaesthetic.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Vehicle (sterile air) group (group 1)

Doses / concentrations 2

Dose / conc.:

30 000 ppm

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

60 000 ppm

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

120 000 ppm

Remarks:

Group 4

No. of animals per sex per dose:

6 males vehicle control and test item groups. 3 males for positive control.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA):
slides prepared from a separate study

Examinations

Tissues and cell types examined:

The proportion of polychromatic erythrocytes was assessed by examination of a total of at
least 1000 erythrocytes per animal and the number of micronucleated normochromatic
erythrocytes was recorded.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
This class of compounds typically have a low level of toxicity and being volatile have the potential for a high level of exposure if accidentally released. The 4-hour acute LC50 for this compound is greater than 105,000 ppm. With most other compounds in this class similar exposure levels were selected for this study.

Slide Preparation:
One femur was dissected out from each animal. The femurs were cleaned of all excess tissue
and blood and the heads of the femurs removed from each bone. The bone marrow of one
femur from each animal was flushed out and pooled in a total volume of 3 mL of filtered
foetal bovine calf serum by aspiration.

The resulting cell suspensions were centrifuged at 1000 rpm (150 g) for 5 minutes and the
supernatant discarded. The final cell pellet was resuspended in a small volume of foetal
bovine calf serum to facilitate smearing in the conventional manner on glass microscope
slides.

The slides were fixed in methanol and allowed to air dry. They were then rinsed in purified
water and stained using an acridine orange solution at 0.0125 mg/mL and stored at room
temperature in the dark until required. Prior to scoring the slides were wet mounted with
coverslips using purified water.

Microscopic Examination:
Slides prepared in a separate study from animals treated with Cyclophosphamide (CPA), a well characterised clastogen, were stained and coded along with the bone marrow smears prepared from this study.

Coded slides were examined by fluorescence microscopy and 4000 polychromatic erythrocytes
per animal were examined for the presence of micronuclei. One smear was examined per
animal, any remaining smears being held temporarily in reserve in case of technical problems
with the first smear.

The proportion of polychromatic erythrocytes was assessed by examination of a total of at
least 1000 erythrocytes per animal and the number of micronucleated normochromatic
erythrocytes was recorded.

Evaluation criteria:

Evaluation and Interpretation of Results:
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
a) None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent vehicle control,
b) There is no dose-related increase at any sampling time when evaluated by an appropriate trend test,
c) All results are inside the distribution of the historical vehicle control data (control limits), and
d) Bone marrow exposure to the test item(s) occurred.

Negative results indicate that, under the test conditions, the test chemical does not produce micronuclei in the polychromatic erythrocytes of the test species.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly
positive if:

a) At least one of the treatment groups exhibits a statistically significant increase in the
frequency of micronucleated polychromatic erythrocytes compared with the concurrent
vehicle control,
b) This increase is dose-related at least at one sampling time when evaluated
with an appropriate trend test, and
c) Any of these results are outside the distribution of the historical vehicle control data (control limits).

When conducting a dose-response analysis, at least three treated dose groups should be
analysed. Statistical tests should use the animal as the experimental unit. Positive results in
the micronucleus test indicate that a test chemical induces micronuclei, which are the result
of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test
species.

There is no requirement for verification of a clear positive or clear negative response.

Statistics:

For the proportion of polychromatic erythrocytes, an asymptotic one-tailed Jonckheere’s test
for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 for a decrease from
control. If significant, then the analysis was carried out on Groups 1 to 3. Exact one-tailed
Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, were also carried out
on Group 1 (control) versus Groups 2, 3, 4 and the positive control.

For incidences of micronucleated polychromatic erythrocytes, an exact one-tailed Linear-by-
Linear association test (Cytel 1995) with “step-down” was used on Groups 1 to 4 for an
increase from control. If significant, then the analysis was carried out on Groups 1 to 3.

Exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, were
also carried out on Group 1 (control) versus Groups 2, 3, 4 and the positive control.

If the exact version of a test could not be calculated (due to the amount of data), then the
asymptotic version was used instead.

Statistical significance was declared at the 5% level for all tests.

The data were received in an Excel document and analyzed using SAS (SAS Institute Inc.
2002) (Jonckheere's and Wilcoxon tests) and StatXact (Cytel 1995) (Linear-by-Linear and
Permutation tests).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Inhalation Analysis:
Mean achieved atmosphere concentrations were 104, 106 and 102% of target for groups 2, 3
and 4 (31100, 63700, 122000 ppm) respectively.


Main Test:
The test was carried out in male animals only. No mortalities were observed throughout the
duration of the test. Please see attachment "Summary of results and statistical analysis - micronucleus phase" for full table detailing result and statistical analysis.

Detailed bodyweights are presented in attachment "Body weights - continued" and clinical signs in attachment "Clinical signs".

Clinical Signs:
Animals were treated with TKN1 at dose levels of 30,000, 60,000 and 120,000 ppm.

Clinical signs including red staining on the head and wet fur were observed in the vehicle control animals.

At 30,000 and 120,000 ppm, clinical signs included red staining on the head and wet fur. At
60,000 ppm, clinical signs included red staining on the head, eyes and nose.

There were no incidences of group mean body weight loss (Day 1 to Day 3. Small incidences
of bodyweight loss between individual weighings were observed in male animals in all
groups.

Micronucleated Polychromatic Erythrocyte Counts (MPCE):
TKN1 did not cause any statistically significant increases in the number of micronucleated
polychromatic erythrocytes in male Crl:CD (SD) rats.

The coded positive control slides prepared demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes.

Micronucleated Normochromatic Erythrocytes (MNCE):
There was no increase in the incidence of micronucleated normochromatic erythrocytes in
male Crl:CD (SD) rats.

Proportion of Polychromatic Erythrocytes (%PCE):
Statistically significant decreases in the proportion of polychromatic erythrocytes (%PCE) were observed in male Crl:CD(SD) rats administered TKN1 at 60,000 and 120,000 ppm, compared to the concurrent vehicle control (p<0.05). There was also a statistically significant trend observed from groups 1 to 4 (p<0.01); this was again significant following the exclusion of group 4 (p<0.05). As the group mean and all individual %PCE values were within the current vehicle historical control range (95% confidence limits), this is considered not to be of biological relevance.

Any other information on results incl. tables

Summary of results and statistical analysis – micronucleus phase

Male Data
Sampling time after 3rd dose		Treatment (ppm)		Proportion of PCE, Group mean % # (SD)		Group mean MPCE/ 4000 PCE # (SD)	Group mean % MPCE #
3 hours		Vehicle (-)		50.6 (0.2)		3.3 (1.0)	0.08
		TKN1 (30000)		50.6 (2.0)		3.0 (1.8)	0.08
		TKN1 (60000)		48.3*^ (1.8)		3.2 (1.5)	0.08
		TKN1 (120000)		49.2**^^ (0.3)		2.8 (2.6)	0.07
		CPA^a (20 mg/kg)		45.3** (1.4)		73.2** (8.3)	1.83
Vehicle	Sterile Air
PCE	Polychromatic erythrocytes
MPCE	Number of micronucleated cells observed per 4000 polychromatic erythrocytes examined
^a	Positive control slides
SD	Standard deviation
#  Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table
Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):
*			p < 0.05		(significant)
**			p < 0.01		(significant)
otherwise			p > 0.05		(not significant)
Results of one-tailed Jonckheere’s test for trend with “step-down”
^			p < 0.05		(significant)
^^			p < 0.01		(significant)
otherwise			p > 0.05		(not significant)

Individual animal data – micronucleus phase

Male Data
Results for individual animals - 3 hour sampling time
Treatment (ppm)		Animal number	Proportion PCE (%)	MPCE (%)	PCE	NCE	MNCE
Vehicle (-)		1	50.8	3 (0.08)	531	515	0
		2	50.9	3 (0.08)	529	511	0
		3	50.6	2 (0.05)	702	686	0
		4	50.7	3 (0.08)	512	497	0
		5	50.2	4 (0.10)	516	511	0
		6	50.6	5 (0.13)	510	497	0
TKN1 (30,000)		7	51.5	2 (0.05)	578	545	0
		8	52.8	6 (0.15)	530	473	0
		9	51.8	2 (0.05)	553	514	0
		10	50.2	1 (0.03)	515	510	0
		11	47.0	4 (0.10)	518	585	0
		12	50.4	3 (0.08)	557	549	0
TKN1 (60,000)		13	47.3	2 (0.05)	597	664	0
		14	46.8	3 (0.08)	518	589	0
		15	49.1	6 (0.15)	504	522	0
		16	46.4	3 (0.08)	546	631	0
		17	51.0	3 (0.08)	520	500	0
		18	49.3	2 (0.05)	589	605	0
TKN1 (120,000)		19	49.3	1 (0.03)	498	513	0
		20	48.7	7 (0.18)	522	550	0
		21	49.2	4 (0.10)	507	523	0
		22	49.6	0 (0.00)	499	508	0
		23	49.0	4 (0.10)	492	512	0
		24	49.6	1 (0.03)	538	547	0
CPAa (20 mg/kg)		1	43.6	71 (1.78)	481	622	0
		2	46.8	84 (2.10)	463	527	0
		3	46.0	61 (1.53)	477	560	0
		4	44.0	75 (1.88)	484	616	0
		5	46.0	75 (1.88)	486	570	0
Vehicle	Sterile Air
PCE	Polychromatic erythrocytes
MPCE	Number of micronucleated cells observed per 4000 polychromatic erythrocytes examined
NCE	Total number of normochromatic erythrocytes examined for micronuclei
MNCE	Number of micronucleated normochromatic erythrocytes observed
^a	Positive control slides

Applicant's summary and conclusion

Conclusions:

It is concluded that TKN1 has not shown any evidence of causing an increase in the induction
of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male
Crl:CD (SD) rats when administered via inhalation in this in vivo test procedure.

Executive summary:

Introduction

This study was designed to assess the potential of TKN1 to induce DNA strand breaks in the liver, kidney, urinary bladder and lungs and also assess the potential induction of micronuclei in the bone marrow cells of Crl:CD (SD) rats.

Animals were treated with TKN1 via the inhalation route on three occasions (6-hour exposure), the second dose being administered approximately 24 hours after the first dose, with the third dose being administered approximately 21 hours after the second dose.
Sampling was performed approximately 3 hours after the completion of the 6-hour exposure period on Day 3.

Dose levels of 30,000, 60,000 and 120,000 ppm were selected for the test. The test was performed using male animals only.

 

Micronucleus Phase

Bone marrow smears were obtained from animals in the vehicle control and in each of the test item groups approximately 3 hours after the completion of the 6-hour exposure period on Day 3.

In addition, slides prepared from a separate study from animals treated with Cyclophosphamide (CPA) a well characterised clastogen, were stained and coded along with the bone marrow smears prepared from this study.

One smear from each animal was examined for the presence of micronuclei in 4000 polychromatic erythrocytes.  The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal.  A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

 

Results/Discussion

The data for the concurrent vehicle control (group mean % polychromatic erythrocytes [%PCE] and % micronucleated polychromatic erythrocytes [%MPCE]) were within the ranges determined by the laboratory historical control data (95% confidence limits), therefore, the performance of the vehicle was consistent with a valid assay. The coded positive control slides prepared from study GY05QJ demonstrated the ability of the analyst to detect increases in micronucleated polychromatic erythrocytes.

There were no significant increases in the group mean MPCE of male rats administered TKN1, compared to the concurrent vehicle control. The individual and group mean %MPCE values from all groups were within the current vehicle historical control range (95% confidence limits).

Statistically significant decreases in the group mean % polychromatic erythrocytes (%PCE) were observed in male rats administered TKN1 at 60,000 and 120,000 ppm, compared to the concurrent vehicle control (p<0.05). There was also a statistically significant trend observed from groups 1 to 4 (p<0.01); this was again significant following the exclusion of group 4 (p<0.05). As the group mean and all individual %PCE values were within the current vehicle historical control range (95% confidence limits), this is considered not to be of biological relevance.

Conclusions

It is concluded that TKN1 has not shown any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male Crl:CD (SD) rats when administered via inhalation in this in vivo test procedure.