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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Comet Assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Apr 2021 to 08 Mar 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

1
Chemical structure
Reference substance name:
octadecasodium ({[2-({2-[bis(hydrogen phosphonatomethyl)amino]ethyl}(hydrogen phosphonatomethyl)amino)ethyl](hydrogen phosphonatomethyl)amino}methyl)phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonatomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate
EC Number:
701-216-4
Molecular formula:
DTPMP-5Na C9H23N3Na5O15P5 DTPMP-6Na C9H22N3Na6O15P5 DTPMP-7Na C9H21N3Na7O15P5
IUPAC Name:
octadecasodium ({[2-({2-[bis(hydrogen phosphonatomethyl)amino]ethyl}(hydrogen phosphonatomethyl)amino)ethyl](hydrogen phosphonatomethyl)amino}methyl)phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonatomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate
Test material form:
solid - liquid: aqueous solution
Remarks:
clear amber liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
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 items. Moreover, historical control background data has been generated with this strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 - 10 weeks
- Weight at study initiation: The mean body weights were for males 268.3 ± 14.9 g and the range 225 – 290g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: Polycarbonate cages (Makrolon MIV type; height 18 cm.) containing sterilized sawdust as bedding material equipped with water bottles. Up to 5 animals of the same sex and same dosing group were housed together.
- Diet (e.g. ad libitum): Pellets (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum, except during designated procedures. Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water. Freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 6 days before the commencement of dosing.

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

IN-LIFE DATES: From: 08/06/21 To: 22/07/2021

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: water (Milli-Q water)
- Justification for choice of vehicle: Product is aqueous solution hence preferred vehicle was water.
- Concentration of test material in vehicle: The test item was dissolved in water. Adjustment was made for specific gravity of the test
item (1.3108 g/mL) and the purity of the test item (24.93% w/w). Taking this into account 3.06 mL of pure test item was considered to be 1g of pure test item. Consequently to achieve for instance a 100 mg/mL solution, 3.06 mL of test item was added to 6.94 mL of vehicle.
- Amount of vehicle (if gavage or dermal): Dose volume: 10 mL/kg
- Type and concentration of dispersant aid (if powder): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Adjustment will be made for specific gravity of the test item (1.3108 g/mL) and the purity of the test item (24.93% w/w). Taking this into account 3.06 mL of pure test item is considered to be 1g of pure test item. The test item will be dissolved or suspended in water. Consequently to achieve for instance a 100 mg/mL solution, 3.06 mL of test item should be added to 6.94 mL of vehicle. Other concentrations can be calculated using this example.
Duration of treatment / exposure:
Two consecutive days
Frequency of treatment:
DRF Study: once daily
Main Study: Twice daily at 0 and 21 hours
Post exposure period:
Animals were sacrificed 3-4 hours after the second dose
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1 - Vehicle (water) only
Dose / conc.:
500 mg/kg bw/day
Remarks:
Group 2 - Treatment group
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group 3 - Treatment group
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
Group 4 - Treatment group
No. of animals per sex per dose:
Main Study: 5 males per dose. Dose range finding study: one group of 3 male and 3 female animals.
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
Ethyl Methane Sulphonate (EMS; CAS No. 62-50-0)
- Justification for choice of positive control(s): As per Table 1 of OECD Test Guideline 489
- Route of administration: oral gavage
- Doses / concentrations: 200 mg ethyl methane sulfonate (EMS)/kg body weight dissolved in physiological saline.

Examinations

Tissues and cell types examined:
Single cell suspensions from liver, duodenum and stomach.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A dose range finding study was performed prior to this experiment where suitable doses were selected. In total 3 male and 3 female animals were dosed with the limit dose of 2000 mg/kg bw for 2 consecutive days. Clinical signs were recorded 2 hours and 21 hours after each dose. No mortalities occurred and no abnormalities were observed, therefore 2000 mg/kg bw was selected as the maximum dose in the main study (also the highest dose required in the current test guideline).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Treatment occurred during two consecutive days. Animals were sacrificed 3-4 hours after the second dose. Sampling occurred after termination of the animals.

Isolation of 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) 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 (40g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359g 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.
The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free). The forestomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA). 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 amount 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. The glandular stomach was then rinsed with mincing buffer incomplete 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 was added immediately before use). The cell suspension was filtered through a 100 μm Cell Strainer 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. The duodenum was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA). 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 to purify the cell suspension and collected in a tube and stored on ice.

DETAILS OF SLIDE PREPARATION: Slides were prepared immediately after the cell suspensions were obtained (finished within 3
hours after necropsy). To the cell suspension, melted low melting point agarose 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 per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 15-27 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

The cells on the slides were overnight (approximately 16-17 h) immersed in pre-chilled lysis solution in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution (1mM EDTA, pH 13) for 20 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 1.0 Volt/cm (liver) or 0.7 Volt/cm (stomach and duodenum). The electrophoresis was performed for 20 (stomach and duodenum) or 30 (liver) minutes under constant cooling (actual temperature 4.0°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5-7 minutes. The slides were subsequently immersed for 5-6 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 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.

METHOD OF ANALYSIS: 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. 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. The occurrence of hedgehogs was scored in all treatment groups and the control. There were no hedgehogs present in any of the slides scored.

HISTOPATHOLOGY: Part of the liver, stomach and duodenum and the tail ID for identification from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was needed.
Evaluation criteria:
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 body weight/day
All results presented in the tables of the report were calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

A test item is considered clearly 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, and
b) The increase is dose-related when evaluated with an appropriate trend test.
c) Any of the results are outside the 95% control limits of the historical negative control data range.

A test item 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 at any sampling time when evaluated with an appropriate trend test,
c) All results are within the 95% control limits of the negative historical control data range
d) direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.
Statistics:
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Remarks:
Water
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
EMS
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw/day for two consecutive days
- Solubility: Not specified
- Clinical signs of toxicity in test animals: In the dose-range finding test, three male and three female animals dosed with 2000 mg test item per kilogram body weight showed no treatment related clinical signs or mortality. No substantial differences in toxicity between sexes and so only males were used in the main study.
- Evidence of cytotoxicity in tissue analysed: Not applicable
- Rationale for exposure: maximum dose for the main test (the highest dose required in the current guideline)

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: The route of exposure and dose levels were appropriate according to the guideline. Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the maximum dose required by the guidelines. Hence, all criteria for an acceptable assay were met.
- Results and Statistical evaluation: No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and stomach cells of test item treated animals compared to the vehicle treated animals. In the stomach, mean Tail Intensities (%) were observed to be slightly higher than the 95% control limits of the distribution of the historical control data for the vehicle control (up to 12.63% versus 11.2%). However, the increase is minor and the results were within the maximum ranges observed in the historical data. Additionally, results were clearly distinct from the positive control (66.67%) and there was no statistically significant increase observed.

The mean Tail Intensity in liver, duodenum and stomach cells of vehicle-treated rats was 2.59 ± 0.52% (mean ± SD), 8.89 ± 0.68% (mean ± SD) and 11.34 ± 1.69% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control for liver and duodenum. The mean Tail Intensity in stomach cells of vehicle-treated rats was slightly above the 95% control limits (11.2%). However, this was considered to have no impact, as the increase is minor (0.14%), within the maximum range observed and it is clearly distinct from the positive control (66.67%). The positive control EMS induced a significant increase and showed a mean Tail Intensity of 88.08 ± 2.64% (mean ± SD), 58.69 ± 2.75% (mean ± SD) and 66.67 ± 1.78% (mean ± SD) in male animals in liver, duodenum and stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.

Applicant's summary and conclusion

Conclusions:
DTPMP (5-7 Na) salt (EC 701-216-4) was tested in a valid in vivo mammalian alkaline comet assay study, conducted according to OECD TG 489 and in compliance with GLP. The substance was not genotoxic in liver, duodenum and stomach cells when sampled approximately 3-4 hours post dosing, in male rats that were dosed via oral gavage for two consecutive days up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines). No clinical signs of toxicity or mortality were observed in the treated animals.