Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

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:

20 December 2021 to 27 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to the guideline study, as a combined OECD 474+489 micronucleus and comet study. All validity criteria were met under both guidelines. The study was conducted under GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 December 2021 to 27 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to the guideline study, as a combined OECD 474+489 micronucleus and comet study. All validity criteria were met under both guidelines. The study was conducted under GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Union No. L142, amendment to EC No. 440/2008, 14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Amber glass bottle, placed in refrigeration (2-8°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Test material concentrations were stirred and treated with ultra-sonic waves to obtain a homogeneous suspension.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Not stated
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Not stated
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): N/A
- Preliminary purification step (if any): N/A
- Final concentration of a dissolved solid, stock liquid or gel: 500, 1000, 2000 mg/kg/day
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: N/A

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han

Details on species / strain selection:

Crl: WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Sex: DRF phase had 3 males and 3 females per group. Main study phase; control and 2000 mg/kg/day had 8 males per group (5 main study animals and 3 TK animals). At 500 or 1000 there were 5 males per group (main study only). Positive control EMS and CP there were 3 males per group (main study only).
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 170.4 - 185.3 (based on group means). Body weights of the rats at the start of the treatment were within 20% of the sex mean.
- Assigned to test groups randomly: Yes.
- Fasting period before study: Nofood
- Housing: Up to 5 animals of the same sex and same dose group were housed together. Housed in polycarbonate cages containing sterilized sawdust as bedding material equipped with water bottles.
- Diet: SM R/M-Z pellets (Sponsor: SSNIFF® Spezialdiäten GmbH, Soest, Germany) available ad libitum. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water, available ad libitum in water bottles.
- Acclimation period: at least 5 days before commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 24 °C
- Humidity: 40 - 70 %:
- Air changes: Ten or more air changes per hour:
- Photoperiod: 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 17 Dec 2021 To: 27 Jan 2022

Route of administration:

oral: gavage

Vehicle:

Corn Oil (Supplier: Fagron, Capelle a/d Ijssel, The Netherlands)
- Justification for choice of solvent/vehicle: Was selected as shown to be suitable in previous in vivo studies using the test material and a solubility test
- Concentration of test material in vehicle: 50 - 200 mg/mL (based on dose level of 500 - 2000 mg/kg/day and dose volume of 10 mL/kg).
- Amount of vehicle (if gavage or dermal): Dose Volume of 10 mL/kg.

Details on exposure:

The test material was suspended in corn oil. Adjustment was made for specific gravity of the vehicle (0.92). In the main study an adjustment was made for specific density of the test material (1.161 g/mL). Test material concentrations were stirred and treated with ultra-sonic waves to obtain a homogeneous suspension. This resulted in yellow translucent suspensions for all formulations. Test material concentrations were dosed within 3 hours after preparation.

Duration of treatment / exposure:

3 consecutive days

Frequency of treatment:

Vehicle and the test material formulations were dosed daily
CP was dosed once daily.
EMS was dosed twice daily.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Negative control group

Dose / conc.:

500 mg/kg bw/day

Remarks:

Test. material

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Test material

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Test material

Dose / conc.:

200 mg/kg bw/day

Remarks:

Postive control - EMS (Comet)

Dose / conc.:

19 mg/kg bw/day

Remarks:

Positive control - CP (micronucleus)

No. of animals per sex per dose:

DRF phase had 3 males and 3 females per group. Main study phase; control and 2000 mg/kg/day had 8 males per group (5 main study animals and 3 TK animals). At 500 or 1000 there were 5 males per group (main study only). Positive control EMS and CP there were 3 males per group (main study only).

Positive control(s):

Alkaline Comet test: Ethyl Methanesulfonate (EMS; CAS no. 62-50-0, Sigma Aldrich, Steinheim, Germany):
-Justification for choice of solvent/vehicle: Based on guideline.
- Concentration of test material in vehicle: 20 mg/mL (based on dose level of 200 mg/kg/day and dose volume of 10 mL/kg) dissolved in physiological saline.
- Amount of vehicle: Dose Volume of 10 mL/kg.

Micronucleus test: Cyclophosphamide (CP; CAS No. 6055-19-2; Sigma Aldrich Chemie GmbH, Steinheim, Germany)
-Justification for choice of solvent/vehicle: Based on guideline.
- Concentration of test material in vehicle: 1.9 mg/mL (based on dose level of 19 mg/kg/day and dose volume of 10 mL/kg) dissolved in physiological saline.
- Amount of vehicle: Dose Volume of 10 mL/kg.

EMS positive control dose twice daily for 3 days via oral gavage.
CP positive control once daily for 3 days via oral gavage.

The positive controls were selected based on the guidelines.

Tissues and cell types examined:

Bone Marrow. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%).

Details of tissue and slide preparation:

Bone marrow was sampled 24 hours after second dosing. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 4 mL of fetal calf serum. The cell suspension was collected and centrifuged at 216 g for 5 min.

The supernatant was removed with a Pasteur pipette. Approximately 500 µl serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, The Netherlands). This staining is based on Giemsa. The dry slides were automatically mounted with a coverslip with an automated coverslipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

Evaluation criteria:

To prevent bias, all slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated. Parts on the slides that contained mast cells that might interfere with the scoring of micronucleated polychromatic erythrocytes were not used for scoring.

Acceptability Criteria of the Micronucleus Test
A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control material induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.
d) The highest test dose is the MTD or 2000 mg/kg/day
ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis.

Statistics:

ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.
A test material is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided,
p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
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.
A test material is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
Negative results indicate that, under the test conditions, the test chemical does not produce micronuclei in immature erythrocytes of the test species.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF THE DOSE FORMULATION ANALYSIS:

Accuracy:
A small response at the retention time of the test material was observed in the chromatograms of the vehicle. It was considered not to derive from the vehicle since a similar response was obtained in the analytical blanks.

The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85%-115%).

Homogeneity:
The dose formulation samples were homogeneous (i.e. RSD ≤ 10%).The mean number of micronucleated polychromatic erythrocytes scored in test material treated groups were compared with the corresponding solvent control group.


RESULTS OF RANGE-FINDING STUDY

- Dose range: 2000 mg/kg bw administered to 3 male and 3 female animals for a total of 3 consecutive days
- Solubility: A solubility test was performed based on visual assessment.
- Clinical signs of toxicity in test animals: None
- Evidence of cytotoxicity in tissue analysed: None
- Rationale for exposure: Maximum according to test guideline
- Harvest times: N/A
- High dose with and without activation: N/A
- Other: N/A

RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay):
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of test material treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the within the 95% control limits of the distribution of the historical negative control database.

Cyclophosphamide, the positive control material, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

- Ratio of PCE/NCE (for Micronucleus assay):
The animals of the groups, which were treated with test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test material on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

- Appropriateness of dose levels and route:
Dose level and route of exposure were selected according to test guideline and guided by a DRF study

Results Summary Table

 

 

Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes

Group	Treatment	Number of Animals	Dose (mg/kg body weight)	Number of micronucleated polychromatic erythrocytes (mean ± S.D.) ^(1)			Ratio polychromatic/ normochromatic erythrocytes (mean ± S.D.) ^(2)
	MALES
1	Vehicle Control	5	0	4.4	±	2.3	0.96	±	0.02
2	Test Material	5	500	5.4	±	2.3	0.87	±	0.15
3	Test Material	5	1000	2.8	±	1.5	0.94	±	0.08
4	Test Material	5	2000	5.8	±	2.3	0.92	±	0.04
6	CP	3	19	68.7	±	13.7^(3)	0.37	±	0.05^(3)

Vehicle control = Corn oil

CP = Cyclophosphamide.

(1)  At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.

(2)  The ratio was determined from at least the first 500 erythrocytes counted.

(3)  Significantly different from corresponding control group (Students t test, P < 0.001).

Conclusions:

The test item was not found to be clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described.

Executive summary:

Study Objective

The objective of this study was to obtain information on the potential genotoxicity of the test material when administered to rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow and by measuring the increase in DNA strand breaks in liver, duodenum and glandular stomach.

 

Study Design

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic materials. Moreover, historical control background data has been generated with this strain.

 

The study procedures described in this report were based on the most recent OECD (29 July 2016) and EC (14 February 2017) guidelines.

 

The test material was a brownish-orange, slightly viscous liquid. The test material was suspended in corn oil.

 

Based on the results of the dose-range finding study, test concentrations of 2000 mg/kg/day for male animals was selected as maximum dose for the main test (the highest dose required in the current guideline). Since there were no substantial differences in toxicity between sexes only males were used in the main study.

 

A small response at the retention time of the test material was observed in the chromatograms of the The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85%-115%). The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%).

 

In the main study male animals were dosed three times by oral gavage with vehicle or with 500, 1000 and 2000 mg test material per kg body weight for three consecutive days. A positive control group for the comet assay was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight and a positive control group for the micronucleus assay was dosed once by oral gavage with 19 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals, with exception of the positive control and TK animals (3 animals per group).

 

In addition, blood for bioanalysis of the test material in plasma was collected from TK animals for the 2000 mg/kg group (highest dose group) and from TK animals for the vehicle control group.

 

In those animals used for bioanalysis (toxicokinetic animals), blood was sampled 0.5, 1, 2, 4 and 6 h after the second dose of either vehicle or the highest concentration of the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.

 

Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated.  Single cell suspensions from liver, glandular stomach and duodenum were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed. Bone marrow smears were prepared for micronucleus analysis.

 

Study Results (Micronucleus)

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test material compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database. Cyclophosphamide, the positive control material, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

The groups that were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test material on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

 

Conclusions

In conclusion, the test material is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Union No. L142, amendment to EC No. 440/2008, 14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

EC Number:

220-482-8

EC Name:

Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

Cas Number:

2781-11-5

Molecular formula:

C9H22NO5P

IUPAC Name:

diethyl {[bis(2-hydroxyethyl)amino]methyl}phosphonate

Test material form:

liquid: viscous

Remarks:

Brownish-orange colour

Details on test material:

- Storage condition of test material: Amber glass bottle, placed in refrigeration (2-8°C)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Amber glass bottle, placed in refrigeration (2-8°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Test material concentrations were stirred and treated with ultra-sonic waves to obtain a homogeneous suspension.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Not stated
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Not stated
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): N/A
- Preliminary purification step (if any): N/A
- Final concentration of a dissolved solid, stock liquid or gel: 500, 1000, 2000 mg/kg/day
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: N/A

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han

Details on species / strain selection:

Crl: WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Sex; DRF phase had 3 males and 3 females per group. Main study phase; control and 2000 mg/kg/day had 8 males per group (5 main study animals and 3 TK animals). At 500 or 1000 there were 5 males per group (main study only). Positive control EMS and CP there were 3 males per group (main study only).
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 170.4 - 185.3 (based on group means). Body weights of the rats at the start of the treatment were within 20% of the sex mean.
- Assigned to test groups randomly: Yes.
- Fasting period before study: Nofood
- Housing: Up to 5 animals of the same sex and same dose group were housed together. Housed in polycarbonate cages containing sterilized sawdust as bedding material equipped with water bottles.
- Diet: SM R/M-Z pellets (Sponsor: SSNIFF® Spezialdiäten GmbH, Soest, Germany) available ad libitum. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water, available ad libitum in water bottles.
- Acclimation period: at least 5 days before commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 24 °C
- Humidity: 40 - 70 %:
- Air changes: Ten or more air changes per hour:
- Photoperiod: 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 17 Dec 2021 To: 27 Jan 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn Oil (Supplier: Fagron, Capelle a/d Ijssel, The Netherlands)
- Justification for choice of solvent/vehicle: Was selected as shown to be suitable in previous in vivo studies using the test material and a solubility test
- Concentration of test material in vehicle: 50 - 200 mg/mL (based on dose level of 500 - 2000 mg/kg/day and dose volume of 10 mL/kg).
- Amount of vehicle (if gavage or dermal): Dose Volume of 10 mL/kg.

Details on exposure:

The test material was suspended in corn oil. Adjustment was made for specific gravity of the vehicle (0.92). In the main study an adjustment was made for specific density of the test material (1.161 g/mL). Test material concentrations were stirred and treated with ultra-sonic waves to obtain a homogeneous suspension. This resulted in yellow translucent suspensions for all formulations. Test material concentrations were dosed within 3 hours after preparation.

Duration of treatment / exposure:

3 consecutive days

Frequency of treatment:

Vehicle and the test material formulations were dosed daily
CP was dosed once daily.
EMS was dosed twice daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Negative control group

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Remarks:

Test. material

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Test material

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Test material

Doses / concentrations 5

Dose / conc.:

200 mg/kg bw/day

Remarks:

Postive control - EMS (Comet)

Doses / concentrations 6

Dose / conc.:

19 mg/kg bw/day

Remarks:

Positive control - CP (micronucleus)

No. of animals per sex per dose:

DRF phase had 3 males and 3 females per group. Main study phase; control and 2000 mg/kg/day had 8 males per group (5 main study animals and 3 TK animals). At 500 or 1000 there were 5 males per group (main study only). Positive control EMS and CP there were 3 males per group (main study only).

Positive control(s):

Alkaline Comet test: Ethyl Methanesulfonate (EMS; CAS no. 62-50-0, Sigma Aldrich, Steinheim, Germany):
-Justification for choice of solvent/vehicle: Based on guideline.
- Concentration of test material in vehicle: 20 mg/mL (based on dose level of 200 mg/kg/day and dose volume of 10 mL/kg) dissolved in physiological saline.
- Amount of vehicle: Dose Volume of 10 mL/kg.

Micronucleus test: Cyclophosphamide (CP; CAS No. 6055-19-2; Sigma Aldrich Chemie GmbH, Steinheim, Germany)
-Justification for choice of solvent/vehicle: Based on guideline.
- Concentration of test material in vehicle: 1.9 mg/mL (based on dose level of 19 mg/kg/day and dose volume of 10 mL/kg) dissolved in physiological saline.
- Amount of vehicle: Dose Volume of 10 mL/kg.

EMS positive control dose twice daily for 3 days via oral gavage.
CP positive control once daily for 3 days via oral gavage.

The positive controls were selected based on the guidelines.

Examinations

Tissues and cell types examined:

Liver, glandular stomach and duodenum cell. Based on the visual scoring of at least 150 cells per tissue per animal.

Details of tissue and slide preparation:

Liver
The isolation method was based on the publication of Hu et al (2002). A portion of
0.6-0.7 gram from the liver was removed and minced thoroughly on aluminum foil in ice. The minced liver tissue was added to 10 mL of collagenase (20 Units/mL; Sigma Aldrich, Zwijndrecht, The Netherlands) dissolved in HBSS (Ca2+- and Mg2+-free) and incubated in a shaking water bath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

Isolation of glandular stomach cells
This isolation method for glandular stomach is based on the JACVAM Comet validation study Uno et al (2015). The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free, Life Technologies, Breda, the Netherlands). The fore-stomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA (Merck, Darmstadt, Germany)). The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore, this layer contains a high number of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis (Uno et al (2015)). The glandular stomach was then rinsed with mincing buffer incomplete (without DMSO) and transferred to a petri-dish containing 10 mL mincing buffer. The glandular stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution, pH 7.5 (DMSO (Merck) was added immediately before use). The cell suspension was filtered through a 100 µm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

Isolation of duodenum
This isolation method for duodenum is based on the JACVAM Comet validation study, Uno et al (2015).
The duodenum was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA without DMSO). The duodenum was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The duodeunum was cut open and the surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper to remove apoptotic cells in the upper cell layer. This layer was discarded. The duodenum was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The duodenum was then scraped multiple times with a cell scraper and the cells are collected in the mincing buffer present in the petri-dish.
The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use). The cell suspension was filtered through a 100 µm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

Preparation of Comet Slides
To the cell suspension, melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 µL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 45 to 47 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

Lysis, Electrophoresis and Staining of the Slides
The cells on the slides were immersed overnight (approximately 16 h) in pre-chilled lysis solution (Trevigen) in the refrigerator (set to maintain 4°C). After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4) in order to remove residual detergent and salts prior to the alkali unwinding step. The slides were then placed in freshly prepared alkaline solution for 20 and 30 minutes, for stomach/duodenum and liver, respectively, at room temperature in the dark to allow DNA unwinding. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 – 1 Volt/cm. The electrophoresis was performed for 20 to 30 minutes under constant cooling (actual temperature 4.0 - 4.5°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in absolut ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

Evaluation criteria:

To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample.
The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.
In addition, the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal in the repeat experiment. The occurrence of hedgehogs was scored in all treatment groups and the control.

The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database.
c) Adequate numbers of cells and doses have been analysed
d) The highest test dose is the MTD or 2000 mg/kg/day
ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis.

Statistics:

ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data .
A test material is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
When all criteria are met, the test chemical is considered to induce DNA strand breakage in the tissues examined within the test system.
A test material is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
When all criteria are met, the test chemical is considered not to induce DNA strand breakage in the tissues examined within the test system.
As the Multiple Sequentially-rejective U-test and Welch t test show that there are statistically significant differences between one or more of the test material groups and the vehicle control group a Linear regression (p < 0.05) was performed to test whether there is a significant trend in the induction.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF THE DOSE FORMULATION ANALYSIS:

Accuracy:
A small response at the retention time of the test material was observed in the chromatograms of the vehicle. It was considered not to derive from the vehicle since a similar response was obtained in the analytical blanks.

The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85%-115%).

Homogeneity:
The dose formulation samples were homogeneous (i.e. RSD ≤ 10%).The mean number of micronucleated polychromatic erythrocytes scored in test material treated groups were compared with the corresponding solvent control group.


RESULTS OF RANGE-FINDING STUDY

- Dose range: 2000 mg/kg bw administered to 3 male and 3 female animals for a total of 3 consecutive days
- Solubility: A solubility test was performed based on visual assessment.
- Clinical signs of toxicity in test animals: None
- Evidence of cytotoxicity in tissue analysed: None
- Rationale for exposure: Maximum according to test guideline
- Harvest times: N/A
- High dose with and without activation: N/A
- Other: N/A

RESULTS OF DEFINITIVE STUDY

- Tail Intenisity (Comet Assay)
A statistical significant increase (p<0.05) in the mean Tail Intensity (%) was observed in duodenum cells of test material treated male animals compared to the vehicle treated animals. The high and intermediate dose groups showed tail intensity increases compared to vehicle-treated animals and in addition a dose related response was observed (significant trend analysis (P<0.0001)). All the results obtained were within the 95% confidence interval of historical control database of the negative controls.

A statistically significant increase (p<0.05) in the mean Tail Intensity (%) was observed in glandular stomach cells of test item treated male animals compared to the vehicle treated animals for the low dose group (500 mg/kg body weight); the intermediate and high dose groups did not show a statistical increase. However, no dose related response was observed (confirmed by a trend analysis) and these values were within the historical control database of the negative controls.

No increase in the mean Tail Intensity (%) was observed in liver cells of test material treated male animals compared to the vehicle treated animals.


Ethyl Methanesulfonate, the positive control material, induced a significant increase and in the mean Tail Intensity in the male animals liver, duodenum and glandular stomach cells. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.

- Appropriateness of dose levels and route:
Dose level and route of exposure were selected according to test guideline and guided by a DRF study

Any other information on results incl. tables

Results Summary Tables

Overview Tail Intensity in Liver Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	2.09	0.35
Test Material 500 mg/kg	1.78	0.28
Test Material 1000 mg/kg	1.89	0.78
Test Material 2000 mg/kg	1.56	0.54
EMS 200 mg/kg	78.04	3.03

Overview Tail Intensity in Duodenum Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	3.90	1.84
Test Material 500 mg/kg	4.81	0.67
Test Material 1000 mg/kg	6.48	1.31
Test Material 2000 mg/kg	9.43	2.27
EMS 200 mg/kg	50.79	3.47

Overview Tail Intensity in Glandular Stomach Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	5.23	0.97
Test Material 500 mg/kg	6.73	1.91
Test Material 1000 mg/kg	5.72	1.04
Test Material 2000 mg/kg	6.22	1.38
EMS 200 mg/kg	52.67	7.18

 

Applicant's summary and conclusion

Conclusions:

The Comet assay undertaken on cells of male rats sampled 3-4 hours post dosing at up to 2000 mg/kg with the test item (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report as found to be valid. The test material is not genotoxic in the Comet assay in liver and glandular stomach cells. The assay was equivocal in duodenum cells as there was a significant dose-response shown however within historical control range of the vehicle control. No morphologic alterations were found which indicate that there was no cytotoxicity present.

Executive summary:

Study Objective

The objective of this study was to obtain information on the potential genotoxicity of the test material when administered to rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow and by measuring the increase in DNA strand breaks in liver, duodenum and glandular stomach.

 

Study Design

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic materials. Moreover, historical control background data has been generated with this strain.

 

The study procedures described in this report were based on the most recent OECD (29 July 2016) and EC (14 February 2017) guidelines.

 

The test material was a brownish-orange, slightly viscous liquid. The test material was suspended in corn oil.

 

Based on the results of the dose-range finding study, test concentrations of 2000 mg/kg/day for male animals was selected as maximum dose for the main test (the highest dose required in the current guideline). Since there were no substantial differences in toxicity between sexes only males were used in the main study.

 

A small response at the retention time of the test material was observed in the chromatograms of the The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85%-115%). The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%).

 

In the main study male animals were dosed three times by oral gavage with vehicle or with 500, 1000 and 2000 mg test material per kg body weight for three consecutive days. A positive control group for the comet assay was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight and a positive control group for the micronucleus assay was dosed once by oral gavage with 19 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals, with exception of the positive control and TK animals (3 animals per group).

 

In addition, blood for bioanalysis of the test material in plasma was collected from TK animals for the 2000 mg/kg group (highest dose group) and from TK animals for the vehicle control group.

 

In those animals used for bioanalysis (toxicokinetic animals), blood was sampled 0.5, 1, 2, 4 and 6 h after the second dose of either vehicle or the highest concentration of the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.

 

Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated.  Single cell suspensions from liver, glandular stomach and duodenum were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed. Bone marrow smears were prepared for micronucleus analysis.

 

Study Results (Comet Assay)

A statistical significant increase (p<0.05) in the mean Tail Intensity (%) was observed in duodenum cells of test material treated male animals compared to the vehicle treated animals. The high and intermediate dose groups showed tail intensity increases compared to vehicle-treated animals and in addition a dose related response was observed (significant trend analysis (P<0.0001)). All the results obtained were within the 95% confidence interval of historical control database of the negative controls.

 

A statistically significant increase (p<0.05) in the mean Tail Intensity (%) was observed in glandular stomach cells of test item treated male animals compared to the vehicle treated animals for the low dose group (500 mg/kg body weight); the intermediate and high dose groups did not show a statistical increase. However, no dose related response was observed (confirmed by a trend analysis) and these values were within the historical control database of the negative controls.

 

No increase in the mean Tail Intensity (%) was observed in liver cells of test material treated male animals compared to the vehicle treated animals.

 

Conclusion

The Comet assay undertaken on cells of male rats sampled 3-4 hours post dosing at up to 2000 mg/kg with the test item (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report as found to be valid. The test material is not genotoxic in the Comet assay in liver and glandular stomach cells. The assay was equivocal in duodenum cells as there was a significant dose-response shown however within historical control range of the vehicle control. No morphologic alterations were found which indicate that there was no cytotoxicity present.