Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 17 December 2013 and 25 June 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
To invetsigate the positive mutagenic result (in the absence of metabolic activation) observed in the CHO HPRT forward mutation assay a Single Cell Gel Electrophoresis (Comet) Assay in the male rat was performed to address concerns for worker safey. It also fills the REACH data requirement.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Draft OECD Guideline for the Testing of Chemicals: In vivo Mammalian Alkaline Comet Assay (2013).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Sufficient male Wistar Han™ (HsdRCCHan™WIST) rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. At the start of the main test the males weighed 181 to 211 g, and were approximately seven to twelve weeks old. Details of the individual animal weights, group means and standard deviations are presented in the attached Table 1 (attached background material). After a minimum acclimatization period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a color coded cage cardThe animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Harlan Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study. The animals were provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The temperature and relative humidity were set to achieve limits of 19 to 25 ºC and 30 to 70% respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The vehicle control was (water for irrigation) supplied by a reputable supplier. Details as follows:Sighting Test:Identification:Water for irrigation* Supplier: Aguettant LtdSupplier’s Lot Number: 3009615 01Harlan Serial Number: V-5541Expiry Date:01 June 2014Storage conditions: Room temperatureMain Experiment:Identification:Water for irrigation*Supplier: Aguettant LtdSupplier’s Lot Number: 3010081Harlan Serial Number: V-5709Expiry Date:01 April 2015Storage Conditions: Room temperature* Referred to as distilled water throughout the report
Details on exposure:
Range-finding Toxicity Test:A range-finding test was performed to find suitable dose levels of the test item following a double oral administration at zero and 20 hours. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. The selection of the maximum dose and the choice of vehicle were based on data from a previous study (41101794), investigating Acute Oral Toxicity in the Rat.A group of rats were dosed orally as follows:Dose Level 2000mg/kg, Concentration 200 mg/mL, Dose Volume 10 mL/kg, Number of Rats 2 All animals were dosed twice 20 hours apart at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of the initial dosing.Animals were observed approximately 1 hour after each dosing and approximately 24 hours after the initial dose. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.Comet Test:Groups each of seven males, was dosed twice with a 20 hour interval via the oral route with the test item at 2000, 1000 or 500 mg/kg. The groups of rats from each dose level were killed using a schedule 1 method approximately 4 hours following the second administration. In addition, two further groups of rats were included in the study; one group (seven male rats) was dosed twice with a 20-hour interval via the oral route with the vehicle alone (distilled water) and a second group (five male rats) was dosed twice orally with 20-hour interval with MNU. MNU is a positive control material that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test. The vehicle and positive controls were killed approximately 4 hours following the second administration. All animals were observed for signs of overt toxicity and death one hour after dosing and immediately prior to termination (24 hours after the initial dose).
Duration of treatment / exposure:
24-hours
Frequency of treatment:
Twice
Post exposure period:
Not applicable
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
The test item concentrations presented throughout the report are calculated based on the active ingredient (85.53%) of the test item and an allowance was made for the water/glycol content when the formulations were prepared.
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
The test item concentrations presented throughout the report are calculated based on the active ingredient (85.53%) of the test item and an allowance was made for the water/glycol content when the formulations were prepared.
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
The test item concentrations presented throughout the report are calculated based on the active ingredient (85.53%) of the test item and an allowance was made for the water/glycol content when the formulations were prepared.
No. of animals per sex per dose:
Comet Test: 7 males per dose group.
Control animals:
yes, concurrent vehicle
Positive control(s):
For the purpose of this study the positive control item N-Nitroso-N-methylurea (MNU) was freshly prepared as required as a solution at the appropriate concentration in distilled water (25 mg/Kg). Five male rats were dosed twice orally with 20-hour interval with MNU. MNU is a positive control material that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test.

Examinations

Tissues and cell types examined:
Tissue Sample RequirementsSamples of liver, bone marrow and glandular stomach were obtained from each animal. Sub-samples of the primary tissues taken from the vehicle control animals and the dose group animals were processed and preserved for possible histopathology investigations. The remaining tissue samples were processed to provide single cell suspensions, providing sufficient cells for scoring, for the Comet Assay as follows:Liver - A small piece of liver was excised (approximately 1 cm3) and washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension. Glandular Stomach – The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension was obtained by scraping the underlying glandular stomach tissue and suspending it in stomach buffer. The resulting cell suspension was filtered through gauze prior to use for the comet slides. Bone Marrow – A femur was dissected from each animal and aspirated with PBS to provide a single cell suspension. The cell suspension was filtered through gauze prior to use for the comet slides. Whole Blood - Samples of whole blood (5 µL per slide) were also processed, fixed and stored but not scored. The above procedures were performed under subdued lighting and the Comet Assay tissues/cells were processed at approximately 4 °C. Blood samples (approximately 1 mL) were taken from the vehicle control animals and the dose group animals into heparinized tubes, centrifuged and the supernatant removed and stored frozen at approximately -20 ºC prior to analysis for presence of the test item.
Details of tissue and slide preparation:
Slide Preparation:Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature prior to the start of the experiment. The slides were labelled for animal number, project number and tissue type prior to use for the comet assay.Once the cell suspensions had been obtained, approximately 30 µL was added to 270 µL of 0.5% low melting point (LMP) agarose and mixed thoroughly and 50 µL of this cell suspension was placed onto one half of a pre-coated slide, 2 slides per animal (4 gels), for each tissue. The agarose was immediately covered with a glass coverslip and kept at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify. All of the slides went through the subsequent processing. Once the LMP agarose had set the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated at approximately 4 °C in the dark overnight.After the lysis phase had been completed the slides were removed from the lysing solution, briefly rinsed with neutralization buffer (0.4M Tris, pH 7.5) and placed onto the platform of a electrophoresis bath, which was filled with chilled electrophoresis buffer (pH >13), until the slide surface was just covered. The slides were then left for approximately 20 minutes to allow the DNA to unwind. When the DNA unwinding period had finished the slides were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for approximately 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis. The aim was to induce sufficient migration of the DNA so that minimal sized comets are produced in the nuclei of vehicle control cells.At the end of the electrophoresis period the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer (0.4M Tris pH 7.5) and allowed to rest for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer performed twice. The slides were then carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry. Once dry the slides were stored prior to scoring. Two of the four processed slides were scored and the remaining slides were stored as backup slides.Scoring:The slides were coded using a computer generated code prior to scoring. To each dry slide, 75 µL of propidium iodide (20 µg/mL) was placed on top of the slide and then overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program, i.e. Comet IV.Two slides for each tissue per animal were scored with a maximum of 75 cells per slide giving an accumulative total of 150 cells per tissue per animal. Care was taken to guarantee that a cell is not scored twice. The slide score data for each tissue was processed using the Excel program provided in Comet IV. Comparisons between the vehicle control group response and that of the test item dose groups would be made. The primary end-point was percentage DNA in the tail (percentage Tail intensity), although other endpoints such as tail moment and length may also be utilized. A positive response is demonstrated when there is clear dose-related change in the defined measurement between the vehicle control and the test item at single sampling time, or a change in the defined measurement in a single dose group at least at a single sampling time. If no marked change in the defined measurement is observed then the test item will be judged as not causing DNA damage in vivo in the tissues evaluated. Although most experiments will be expected to give clear negative or positive results in rare cases the data set will preclude making a definite judgment. This may require the scoring of additional slides to increase the number of cells and, therefore, add more power to the data. If this does not resolve the issue then the result will be given as equivocal or questionable, and may require the histopathological assessment of the tissues see if cell toxicity may be the causative agent rather than any genotoxic mechanism.Histopathology:Samples of the above primary tissues taken from the vehicle control animals and the dose group animals were preserved in buffered 10% formalin and stored for possible histopathology processing. Plasma Analysis:Frozen blood serum/plasma samples were analyzed by the Department of Analytical Services for triethanolamine concentration by High Performance Liquid Chromatography using Mass Spectrometry (HPLC MS). Validation of the analytical method was performed by spiking in known amounts of the suspected hydrolysis product, triethanolamine, into control plasma and determining accuracy and precision data. Full details of the analytical method used is documented in the original data, approved prior to use and detailed in the Plasma Analysis Report (Attached Appendix 1 - attached background material).
Evaluation criteria:
Refer to 'scoring' section above.
Statistics:
No statistical analysis was performed

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:
Range-Finding Toxicity TestThe mortality data are summarized as follows: In animals dosed with test item there were no premature deaths, and no clinical signs observed. Based on the above data the maximum recommended dose (MRD) of the test item, 2000 mg/kg, was selected for use in the main test, with 1000 and 500 mg/kg as the lower dose levels. Comet Assay:Mortality Data and Clinical Observations:There were no premature deaths seen in any of the test item dose groups. No clinical signs were observed in animals dosed with the test item at any of the dose levels or time points. Evaluation of Comet Assay Slides:A table of the combined group data for each tissue scored for the Comet Assay; glandular stomach, liver and bone marrow, is given in the attached Table 2. Individual animal data for each tissue are presented in the attached Tables 3 to 5 with the means, medians and standard deviations calculated from the individual animal data and group data calculated from the combined individual animal data.The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control range. The positive control group induced marked increases in percentage tail intensity in all tissues analyzed which were consistent with the laboratory historical positive control range. The test method itself was therefore operating as expected and was considered to be valid under the conditions of the test.There was no marked increase in percentage tail intensity for any of the test item dose levels in any of the tissues analyzed when compared to the vehicle control. Confirming that the test item did not induce DNA damage in the tissue investigated under the conditions of the test.There was no marked increase in hedgehog frequency for any of the test item dose levels in any of the tissues investigated. The hedgehog frequency data for each tissue is presented in the attached Tables 6 to 8. Tables 2-8 in attached background material.

Any other information on results incl. tables

Discussion

The test item did not demonstrate any increases in percentage tail intensity over the vehicle controls in any of the tissues investigated. There was also no dose related increase in the group median values for any of the tissues investigated indicating no shift in the percentage tail intensity between test item groups. 

 

The percentage tail intensity for the vehicle control in the glandular stomach was higher than that observed in the dose groups but this considered to be as a result of high values in three animals (animals 3, 4 and 7) and confirmed by the high standard deviations in these animals. The gastro-epithelial lining has a high rate of proliferation and therefore cell destruction and it is considered that in these animals a higher proportion of dying or damaged cells were sampled resulting in the higher overall percentage tail intensity values. Although the increase was not so marked in the 500 mg/Kg dose level there were also two animals with elevated percentage tail intensities (animals 32 and 33) which increased the overall percentage tail intensity for the group. However, the overall group values for the vehicle control and the 500 mg/Kg dose level were well below the response seen in the positive control group, were within the laboratory historical range for a vehicle control and were not considered to be part of a positive response.

Low percentage tail intensity values were achieved in the vehicle control and the test item dose levels in the liver and bone marrow indicating that there was consistently little damage in these tissues at the test item dose levels tested. Although some of the group and individual median values reported were zero these were rounded values and reflected very low levels of migration rather than no migration or negative migration.

The hedgehog frequency was seen to be higher in all the positive control groups compared to the vehicle control and the dose groups, possibly as a result of cytotoxicity, but did not show any dose related increases in the test item dose groups for any of the tissues investigated. The glandular stomach demonstrated higher levels of hedgehog frequency generally but this is considered to be due in part to the high turnover of cells in the glandular stomach with high numbers of dying cells being constantly present. 

Analysis of the blood serum from the animals of the 2000 mg/Kg dose group demonstrated the presence of triethanolamine indicating that the test item had been absorbed into the blood stream and therefore the liver and bone marrow will have been exposed. The glandular stomach will have been exposed to the test item as it was dosed orally. It is assumed that with evidence of test item absorption in the highest dose group, that the lower dose groups will have also been absorbed.

Applicant's summary and conclusion

Conclusions:
The test item did not induce any increases in the percentage tail intensity values of the liver, glandular stomach or bone marrow. The test item was considered to be non-genotoxic to the rat tissue investigated in vivo.
Executive summary:

Introduction

This test method has been designed using the recommendations of the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington DC in 1999, as described by Ticeet al.(2000). At the time a method for this assay had not yet been adopted by the OECD, active discussions were in progress to develop an approved OECD Method. The test method was designed to be compatible with the procedures indicated in the Draft OECD Guideline for the Testing of Chemicals: In vivo Mammalian Alkaline Comet Assay (2013). 

The method detects evidence of primary DNA damage, as opposed to any of the potential resultant biological effects (e.g. micronuclei, mutations, structural chromosomal aberrations). The assessment of damage to the DNA in single cells is made using an alkaline gel electrophoresis method. The method is capable of detecting DNA single strand breaks (SSB), and double strand breaks (DSB) and alkali labile sites (ALS). Under modified conditions DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites can be detected. Advantages of the method over other genotoxicity tests include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample and its flexibility including adaptation to multiple target tissues.

 

The primary target tissues of this assay were liver, glandular stomach and bone marrow.

Methods

The maximum dose selected for the range-finding test was the maximum recommended dose of 2000 mg/kg. The selection of the maximum dose and the vehicle was based on data from a previous study (41101794), investigating Acute Oral Toxicity in the Rat. The Comet assay main test was conducted at the maximum recommended dose (MRD) 2000 mg/kg with 1000 mg/kg and 500 mg/kg as the lower dose levels. Animals were killed 4 hours after the second dose administration, the stomach and liver tissues were processed and the bone marrow extracted, the slides were then prepared and processed prior to scoring for the presence of Comets. Comet slides were also prepared from blood sampled but not scored. At the request of the Sponsor blood samples were taken from the vehicle control and test item dose groups and stored for possible future analysis. The samples from the vehicle control group and the high dose group (2000 mg/kg) were analysed for the presence of the test item hydrolysis product, triethanolamine.

Further groups of rats were given an oral dose of distilled water (seven rats) or N-Nitroso-N-methylurea (MNU) (5 rats), to serve as vehicle and positive controls respectively.

Results

There was no evidence of an increase in the percentage tail intensity values in the liver, glandular stomach or bone marrow in animals dosed with the test item when compared to the concurrent vehicle control group.

 

The positive control material produced a marked increase in the percentage tail intensity in the liver, glandular stomach and bone marrow, indicating that the test method was working as expected.

 

Conclusion

The test item did not induce any increases in the percentage tail intensity values in any of the tissues investigated. The test item was considered to be non-genotoxic to the rat tissue investigatedin vivo.