Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

single cell gel electrophoresis (comet) assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 April 2015 to 05 June 2015

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

yes

Remarks:

unathorised necropsies performed during range-finding study with no impact on results or integrity of study (see below)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

- Appearance/physical state: Black solid
- Storage conditions: Room temperature, protected from light under nitrogen

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

ANIMALS AND ANIMAL HUSBANDRY
- Sufficient male Wistar Han (HsdRCCHan WIST) rats were supplied by Envigo RMS (UK) Limited.
- At the start of the main test the males weighed 184.2 to 216.9 g, and were approximately eight to ten weeks old.
- Details of the individual animal weights, group means and standard deviations are presented in Table 1 (attached).
- After a minimum acclimatisation 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 colour coded cage card.
- The animals were housed in groups of up to four in solid-floor polypropylene cages with wood-flake bedding.
- Free access to mains drinking water and food (Envigo Teklad 2014C Global Certified Rodent Diet supplied by Envigo RMS Limited, Oxon, UK) was allowed throughout the study. Representative analyses of food and water quality are retained in the laboratory archive.
- The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 %, respectively.
- The rate of air exchange was approximately fifteen changes per hour.
- Lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

VEHICLE CONTROL
- Identification: Arachis oil
- Serial number: V-5957
- Expiry date: 13 June 2016
- Storage conditions: Room temperature

Details on exposure:

TEST ITEM PREPARATION
- The test item was freshly prepared as required as a solution at the appropriate concentration in arachis oil.
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulations.
- The test item was formulated within two hours of it being applied to the test system and it was assumed that the formulations were stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

PREPARATION OF POSITIVE CONTROL
- the positive control item, N-Nitroso-N-methylurea, was freshly prepared as required as a solution at the appropriate concentration in distilled water (Laboratoire Aguettant 3011010).

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 of2000 mg/kg.
- Only male rats were used for the study since there was no marked difference in toxicity demonstrated between the sexes in a previous mouse micronucleus study.
- Groups of male rats were dosed as shown in the table below.
- 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 1 hour after each dosing and immediately prior to termination.
- Any deaths and evidence of overt toxicity were recorded at each observation.

COMET TEST
- Experimental design is summarised in the table below.
- The groups of rats from each dose level were killed 24 hours following the initial treatment.
- Groups each of seven male rats were dosed twice with a 20 hour interval via the oral route with the test item at 1200, 600 or 300 mg/kg.
- The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) 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 (arachis oil) and a second group (five male rats) was dosed twice orally with 20-hour interval with N-Nitroso-N-methylurea (MNU) to act as the positive control. 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 control groups of rats were killed by humane euthanasia (carbon dioxide asphyxiation) 24 hours after the start of the test.
- All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Two doses (20 hours apart)

Post exposure period:

Not applicable

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 200 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

600 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Seven

Control animals:

yes, concurrent vehicle

Positive control(s):

POSITIVE CONTROL MATERIAL
- Identification: N-Nitroso-N-methylurea
- Lot number: P102-01944
- Serial number: R-6206
- Purity: 90 %
- Expiry date: 12 April 2017
- Storage conditions: Approximately 4 °C in the dark

Examinations

Tissues and cell types examined:

- The primary target tissues investigated in this study were liver and glandular stomach.

Details of tissue and slide preparation:

TISSUE SAMPLE REQUIREMENTS
- Humane euthanasia was performed on the animals at the end of the exposure period, using a method that did not affect the integrity of the required tissues. Samples of liver 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. Assessment of cytotoxicity by histopathology is conducted if the results from the Comet assay, or other observations, suggest cytotoxicity may be confounding the interpretation of the Comet assay.
- The remaining tissue samples were processed to provide single cell suspensions, providing sufficient cells for scoring, for the Comet Assay.
- 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 possible 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.
- The above procedures were performed under subdued lighting and the Comet Assay tissues/cells were processed at approximately 4 °C.

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, study 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, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets and two were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension 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.
- After the LMP agarose had set the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark for at least 1 hour.
- After the lysis phase had been completed the slides were removed from the lysing solution, briefly rinsed with neutralisation buffer and placed onto the platform of a electrophoresis bath, which was filled with chilled electrophoresis buffer, 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 neutralisation buffer and allowed to rest for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralisation 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 slide gels were scored and the remaining slides were stored as backup slides.

HISTOPATHOLOGY
- Samples of the above primary tissues from all animals were preserved in buffered 10% formalin.

TREATMENT OF RESULTS
- Individual animal data and grouped data is presented. The median % DNA for each slide was determined and the mean of the median value calculated for each animal. The mean of the individual animal means is calculated to give a group mean. Alternative methods for calculating and presenting the data may be utilised if considered appropriate.
- When there is no indication of any increase in % Tail Intensity at any dose level then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual dose level, using an appropriate statistical method.

ACCEPTABILITY CRITERIA
- The following criteria are used to determine a valid assay:
(i) The concurrent negative control is comparable with the laboratory historical negative control range.
(ii) The positive controls induce responses that are comparable with those in the laboratory positive control range.
(iii) Adequate numbers of cells and doses have been analysed.
(iv) The highest dose level selected meets the requirements of the guideline and the study plan.

Evaluation criteria:

EVALUATION AND INTERPRETION OF RESULTS
- Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly negative if:
(i) None of the test concentrations exhibits a significant increase compared with the concurrent negative control.
(ii) There is no evidence of a dose-related response.
(iii) The results are within the laboratory historical vehicle control range.
(iv) There is evidence, direct or indirect, to demonstrate exposure or toxicity to the target tissue has been achieved.
- The test item is then considered unable to induce DNA strand breakage in the tissues studied in the test system.
- Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
(i) At least one of the test doses exhibits a statistically significant increase compared to the concurrent negative control.
(ii) The response is considered to be dose related.
(iii) The results are substantially outside the laboratory historical vehicle control range.
- The test item can be considered to induce DNA strand breakage in a particular tissue if all three conditions are met.
- There is no requirement for verification of a clearly positive or negative response.
- Although most experiments will be expected to give clear negative or positive results in rare cases the data set will preclude making a definite judgement. 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 to see if cell toxicity may be the causative agent rather than any genotoxic mechanism.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING TOXICITY TEST
- Mortality data are summarised in the table below.
- In animals dosed with test item there was a premature death at 2000 mg/kg after the second dosing. Clinical signs were also observed at termination at 1200, 1500 and 2000mg/kg and these included hunched posture, lethargy, splayed gait, pilo-erection, diarrhoea, staining around the mouth and decreased respiratory rate. There were no clinical signs seen in the animals dosed with the test item at 1000 mg/kg.
- Based on the above data the maximum tolerated dose (MTD) of the test item, 1200 mg/kg, was selected for use in the main test, with 600 and 300 mg/kg as the lower dose levels. The clinical observations noted at termination of the animals (hunched posture and lethargy) were considered to be acceptable.

COMET ASSAY – MORTALITY DATA AND CLINICAL OBSERVATIONS
- There were no premature deaths seen in any of the test item dose groups.
- Clinical signs were observed in animals dosed with the test item at and above 600 mg/kg and these included
hunched posture and pilo-erection.

EVALUATION OF COMET ASSAY SLIDES
- A summary of the combined group data for each tissue scored for the Cornet Assay (glandular
stomach and liver) is presented in the table below.
- Individual animal data for each tissue are presented in Tables 2 to 11 (attached) with the means, medians and standard deviations calculated from the individual animal data and the mean of median percentage tail intensity group data.
- Historical control data for % tail intensity are presented in Appendix 1 (attached).
- The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control range. The positive control material (MNU) produced a marked increase in the percentage tail intensity and median % tail intensity in the liver and glandular stomach. 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 the glandular stomach or the liver tissues when compared to the vehicle control, confirming the test item did not induce DNA damage in the glandular stomach or the liver 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 included in Tables 2 to 11 (attached).

Any other information on results incl. tables

MORTALITY IN THE RANGE-FINDING TOXICITY TEST

Dose level (mg/kg)	Sex	Number of animals treated	Route	Deaths on day 0	Deaths on day 1
1000	Male	2	Oral	0	0
2000	Male	2	Oral	0	1
1500	Male	2	Oral	0	0
1200	Male	2	Oral	0	0
1200	Male	2	Oral	0	0

COMET TEST –-GLANDULAR STOMACH RESULTS

Dose group	Group mean % hedgehogs	Group mean % tail intensity	Group mean of mean of median % tail intensity per animal
Vehicle	2.75	2.67	1.12
300 mg/kg	4.21	2.57	1.06
600 mg/kg	3.28	1.44	0.36
1200 mg/kg	3.34	1.51	0.33
Positive control (MNU)	6.74	31.37	30.11

 

COMET TEST – LIVER RESULTS

Dose group	Group mean % hedgehogs	Group mean % tail intensity	Group mean of mean of median % tail intensity per animal
Vehicle	0.14	0.52	0.02
300 mg/kg	0.21	0.68	0.03
600 mg/kg	0.00	0.44	0.01
1200 mg/kg	0.21	0.45	0.03
Positive control (MNU)	1.16	21.38	21.28

Applicant's summary and conclusion

Conclusions:

The test item did not induce any increases in the % tail intensity values in either the glandular stomach or liver tissue investigated. The test item was considered to be non-genotoxic to the rat tissue investigated in vivo.

Executive summary:

GUIDELINE

The investigation was performed in accordance with OECD Guideline for the Testing of Chemicals No 489 “In vivo Mammalian Alkaline Comet Assay”, adopted 26 September 2014. The Comet Assay has been designed using the recommendations of the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington DC 1999, as described by Ticeetal.,2000. The primary target tissues investigated in this study were liver and glandular stomach.

 

METHODS

A range-finding test was performed to find suitable dose levels of the test item. The study was performed using only male rats, based on the toxicity response seen in the Mouse Micronucleus Test. Groups, each of seven rats, were dosed twice at 0 and 20 hours with the test item via the oral route. The Comet assay main test was conducted at the maximum tolerated dose (MTD) 1200 mg/kg with 600 mg/kg and 300 mg/kg as the lower dose levels. Animals were killed 4 hours after the second dose administration, the glandular stomach and liver tissues processed and the slides were then prepared and processed prior to scoring for the presence of Comets. Further groups of rats were given a double oral dose of arachis oil (seven rats) or N-Nitroso-N-methylurea (five rats), to serve as vehicle and positive controls respectively.

 

RESULTS

The presence of clinical signs indicated that systemic absorption had occurred. There was no evidence of an increase in the % tail intensity values in animals dosed with the test item dose groups when compared to the concurrent vehicle control group. The positive control material produced a marked increase in the % tail intensity value in all tissues scored, indicating that the test method was working as expected.

 

CONCLUSION

The test item did not induce any increases in the % tail intensity values in either the glandular stomach or liver tissue investigated. The test item was considered to be non-genotoxic to the rat tissue investigated in vivo.