3-aminopropyldiethylamine

Genetic toxicity: in vivo

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:

Experimental Start Date (Experiment 1): 03 October 2019
Experimental Completion Date (Experiment 2): 15 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

3-aminopropyldiethylamine

EC Number:

203-236-4

EC Name:

3-aminopropyldiethylamine

Cas Number:

104-78-9

Molecular formula:

C7H18N2

IUPAC Name:

(3-aminopropyl)diethylamine

Test material form:

liquid

Details on test material:

Appearance: colorless liquid
Storage condition: At room temperature in transparent glass flasks in an opaque plastic flask

Specific details on test material used for the study:

Diethylaminopropylamine, also known as 3-aminopropyldiethylamine; 3 (Diethylamino)propylamine; DEAPA (CAS Number 104-78-9), batch number I133306836 was a colourless liquid. It was received on 01 July 2019 and stored at 15 25¿C, protected from light. Purity was stated as 99.83% and the expiry date was given as 12 May 2021, see Certificate of Analysis. The test article information and certificate of analysis are considered an adequate description of the characterisation, purity and stability of the test article.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was selected as there is a large volume of background data in this species.

Sex:

male

Details on test animals or test system and environmental conditions:

Species, Strain and Supplier
51 male and 9 female young adult out-bred Sprague Dawley rats (Crl:CD(SD)) were obtained from Charles River (UK) Ltd., Margate, UK.

Animals not dosed in this study were transferred to Covance Laboratories Ltd. stock.

Specification
3 male and 6 female animals were dosed during the Range-Finder Experiment. They were approximately 7 to 8 weeks old and 247-285 g (males) or 184-201 g (females) on the first day of dosing.

27 male animals were dosed during Main Experiment 1. They were approximately 7 to 8 weeks old and 240-266 g on the first day of dosing.

15 male animals were dosed during Main Experiment 2. They were approximately 7 to 8 weeks old and 244-295 g on the first day of dosing.

Environment
Animals were housed in wire topped, solid bottomed cages, with three animals of the same sex per cage.

The animals were housed in rooms air-conditioned to provide 15-20 air changes/hour and set to maintain temperature and relative humidity in the range 19-25¿C and 40 70%, respectively.

Fluorescent lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800) and 12 hours dark. The animals were routinely kept under these conditions except for short periods of time where experimental procedures dictated otherwise.

Diet, Water, Bedding and Environmental Enrichment
Throughout the study the animals had ad libitum access to 5LF2 EU Rodent Diet. Each batch of diet was analysed for specific constituents and contaminants.

Mains water was provided ad libitum via water bottles. The water supply was periodically analysed for specific contaminants.

Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd., Manchester). The bedding was analysed for specific contaminants.

In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and rodent retreats.

No contaminants were present in any of the above at levels that might interfere with achieving the objective of the study. Results of any analyses performed are held centrally at Covance Laboratories.

Allocation to Treatment Group
On arrival, animals of the same sex were randomly allocated to cages. Range-Finder were allocated to groups of three and Main Experiment animals were randomised to groups of six (three for the positive control).

Checks were made to ensure the weight variation of Main Experiment animals prior to dosing was minimal and did not exceed ±20% of the mean weight.

Identification of the Test System
The animals were individually identified by uniquely numbered tail mark (Range Finder) or subcutaneous electronic transponder (Main Experiment). Cages were appropriately identified (using a colour-coded procedure) with study information including study number, study type, start date, number and sex of animals, together with a description of the dose level and proposed time of necropsy.

Acclimatisation and Health Procedures
All animals were given a clinical inspection for ill health on arrival. They were acclimatised for at least 5 days and a health inspection was performed before the start of dosing to ensure their suitability for the study.

Experimental Observations
Health Monitoring
All animals were examined at the beginning and the end (nominal) of the working day to ensure that they were in good health and displayed no signs of overt toxicity. Decedents were subjected to necropsy and findings were recorded in the raw data but have not been reported.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Reverse osmosis water

Details on exposure:

Test Article Formulation
Preparation
Formulations were prepared once per experiment by formulating Diethylaminopropylamine in Reverse Osmosis water (pH adjusted to 8.0±0.5) as follows:
The test article was weighed into a pre-labelled container. Approximately 80% of the final volume of vehicle was added to the formulation bottle and the formulation mixed using a magnetic stirrer until homogenous. The pH of the formulations was measured and adjusted to pH8±0.5 using HCl, as required, and made up to the final volume. The final pH was recorded.

Duration of treatment / exposure:

The test article and vehicle control were given as two administrations, at 0 and 21 hours; the EMS positive control was administered once only at 21 hours. All animals were sampled at 24 hours.

Frequency of treatment:

0 and 21 hours

Post exposure period:

Range-Finder Experiment
Day 1: Prior to dose, immediate, 0.5, 1, 2 and 4-6 hours post dose
Day 2: Prior to dose, immediate 0.5, 1, 2 and 4-6 hours post dose

Main Experiments 1 and 2
Day 1: Prior to dose, immediate, 1, 2 and 4 hours post dose
Day 2 2 Prior to dose, immediate and prior to necropsy

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Experiment 1 - Group 1: vehicle control

Doses / concentrations 2

Dose / conc.:

437 mg/kg bw/day (actual dose received)

Remarks:

Experiment 1 - Group 2: Due to lower than expected analytical results, the actual dose levels administered have been reported

Doses / concentrations 3

Dose / conc.:

875 mg/kg bw/day (actual dose received)

Remarks:

Experiment 1 - Group 3: Due to lower than expected analytical results, the actual dose levels administered have been reported

Doses / concentrations 4

Dose / conc.:

1 575 mg/kg bw/day (actual dose received)

Remarks:

Experiment 1 - Group 4: Due to lower than expected analytical results, the actual dose levels administered have been reported

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Experiment 2: vehicle control

Doses / concentrations 6

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

Experiment 2: diethylaminopropylamine

No. of animals per sex per dose:

Main experiments: 6 males per dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate 200 mg/kg, single oral administration at 21 hours (Day 2).

Examinations

Tissues and cell types examined:

Diethylaminopropylamine was tested for its potential to induce DNA strand breaks in the liver, stomach and duodenum of treated rats. The gonad was also sampled but not analysed.
(Day 2, equivalent to 24 hours)

The liver was examined as it was expected to be highly exposed to both the test article and any metabolites of the test article.
Based on ECHA guidance, both the duodenum and stomach were selected for comet investigation as key sites of contact following oral gavage administration.

Details of tissue and slide preparation:

Justification for Dose Selection
The following information on the in vivo toxicity of Diethylaminopropylamine was provided:

A 2-week dose range finding study has been previously conducted in groups of male and five female Sprague Dawley rats with Diethylaminopropylamine at 100, 300 or 1000 mg/kg/day. No treatment-related effects were observed in animals dosed at 100 and 300 mg/kg/day. At 1000 mg/kg/day, only excessive salivation (which is not considered to be an adverse sign of toxicity) was observed in 1/5 males from Day 7 and 4/5 females from Days 2, 3, 8 or 11. There were no notable losses in animal bodyweight (CiToxLAB Study Number 42759 TSR).

Based on this information an initial dose of 2000 mg/kg was administered in a Range Finder Experiment. An additional dose of 1400 mg/kg/day was tested in females only until an estimate of the MTD was determined for each sex (OECD, 2016).

From the results of the Range-Finder Experiment dose levels of 500, 1000 or 2000 mg/kg/day Diethylaminopropylamine (equivalent to 25% of the maximum dose, 50% of the maximum dose and the regulatory maximum dose, respectively) were tested in Main Experiment 1. Following consistent low recovery from the formulation analysis data actual dose levels tested were 437, 875 and 1575 mg/kg/day were administered. Therefore, it was decided that the Main Experiment would be repeated at the high dose of 2000 mg/kg/day only (designated Main Experiment 2).

Both male and female animals were used in the Range-Finder Experiment. However, as there were no substantial gender differences in toxicity (considered to be a difference in MTD of 2-fold or greater), and the Sponsor confirmed there were no data available to suggest differences in metabolism or bioavailability, or gender specific human exposure, Main Experiments 1 and 2 were conducted in male animals only.


For histopathology, a sample of liver, stomach and duodenum from vehicle control and test article treated animals only were removed, immediately preserved in neutral buffered formalin and stored at room temperature. No histopathology samples were preserved for the positive control animals.

Histopathology
Preserved liver, stomach and duodenum samples were embedded in wax blocks and sectioned at 5 µm nominal. Slides were stained with haematoxylin and eosin and examined by the Study Pathologist.

Preparation of Cell Suspensions
The comet liver samples were washed thoroughly in Merchants solution and placed in fresh buffer. The samples were cut into small pieces in Merchants solution and the pieces of liver were then pushed through bolting cloth (pore size of 150 µm) with approximately 4 mL of Merchants solution to produce single cell suspensions.

The comet stomach samples were washed in Merchants solution and then incubated on ice for 15 minutes, covered in fresh Merchants solution. After incubation the stomach samples were removed and placed in 200 µL of fresh Merchants solution. Cells were gently scraped from the inside surface of the stomach using the back of a scalpel blade to produce single cell suspensions.

The comet duodenum samples were washed thoroughly in Merchants solution and placed into fresh buffer. Each sample was vortexed in Merchants solution for approximately 15 seconds. The tissue was removed from the Merchants solution and the inner surface gently scraped (released material discarded) using the back of a scalpel blade. The tissue was vortexed in Merchants solution for a further 15 seconds prior to gently scraping the inside of the duodenum with the back of a scalpel blade.

The comet gonad samples were washed in Merchants solution, an incision was made along the length of each sample and the contents gently squeezed out of the membrane. The membrane was discarded. The remaining material cut into small pieces and pushed through bolting cloth (pore size of 150 µm) with approximately 10 mL of Merchants solution to produce single cell suspensions.

All cell suspensions were held on ice prior to slide preparation.

Slide Preparation
Three slides, labelled ‘A’, ‘B’ and ‘C’ were prepared per single cell suspension per tissue. Slides were labelled with the study number, appropriate animal tag number and tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 µL of each single cell suspension was added to 400 µL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 µL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on ice.

Cell Lysis
Once gelled the coverslips were removed and all slides placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH adjusted to pH 10 with NaOH, 1% Triton X 100, 10% DMSO) overnight at 2-8°C, protected from light.

Unwinding and Electrophoresis
Following lysis, slides were washed in purified water for 5 minutes, transferred to electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) at 2-8°C and the DNA unwound for 20 minutes (stomach and duodenum) or 30 minutes (liver and gonad). At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 20 minutes (stomach and duodenum) or 40 minutes (liver and gonad). As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.

Neutralisation
At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0 (3 x 5 minute washes). After neutralisation the slides were dried and stored at room temperature prior to scoring.

Staining
Prior to scoring, the slides were stained with 100 µL of 2 µg/mL ethidium bromide and coverslipped.

Slide Analysis
Scoring was carried out using fluorescence microscopy at an appropriate magnification and with suitable filters.

A slide from a vehicle and positive control animal were checked for quality and/or response prior to analysis. All slides were allocated a random code and analysed by an individual not connected with soring of the study.

All available animals per group were analysed.

Measurements of tail intensity (%DNA in tail) were obtained from 150 cells/animal/tissue. In general this was evenly split over three slides.

The number of ‘hedgehogs’ (a morphology indicative of highly damaged cells often associated with severe cytotoxicity, necrosis or apoptosis) observed during comet scoring was recorded for each slide. To avoid the risk of false positive results ‘hedgehogs’ were not used for comet analysis. Each slide was scanned starting to the left of the centre of the slide.

The following criteria were used for analysis of slides:
1. Only clearly defined non overlapping cells were scored
2. Hedgehogs were not scored
3. Cells with unusual staining artefacts were not scored.

Slides prepared from the left gonad were prepared but were only to be scored and evaluated if required (if liver, stomach or duodenum elicited a positive result). These slides were subsequently not required for analysis. All comet slides were retained until report finalisation; at this time the slides were discarded with SD approval. Due to the nature of the slides, long term storage is not recommended as comet integrity cannot be assured.

Evaluation criteria:

Evaluation Criteria
For valid data, the test article was considered to induce DNA damage if:

1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control

2. The increase was dose related in any tissue

3. The increase exceeded the laboratory’s historical control data for that tissue.

The test article was considered positive in this assay if all of the above criteria were met.

The test article was considered negative in this assay if none of the above criteria were met and target tissue exposure was confirmed.

Results which only partially satisfied the criteria were dealt with on a case-by-case basis.
Biological relevance was taken into account, for example comparison of the response against the historical control data and consistency of response within and between dose levels and any additional experiments.

Statistics:

After completion of microscopic analysis and decoding of the data the percentage tail intensity (i.e. %DNA in the tail) was calculated.

Data were treated as follows:

1. The median value per slide was calculated
2. The mean of the slide medians was calculated to give the mean animal value
3. The mean of the animal means and standard error of the mean was calculated for each group.

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

Data was analysed using one-way analysis of variance (ANOVA) with the fixed factor for treatment group. The positive control group was excluded from this analysis. Levene’s test was used to test for equality of variances among groups. This showed no evidence of heterogeneity (P>0.01). Comparisons between each treated group and control were made using Dunnett’s test. The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p value are reported. In addition, a linear contrast was used to test for an increasing dose response.

The positive control groups were compared to the control group using a two-sample t test. Levene’s test was used to test for equality of variances between the groups. This showed no evidence of heterogeneity (P>0.01). In both Experiments 1 and 2, the test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p-value are reported.

Results and discussion

Test results

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

under the conditions of this comet assay, Diethylaminopropylamine did not induce biologically relevant increases in DNA strand breaks in the liver, stomach or duodenum when tested up to 2000 mg/kg/day (the max recommended dose for in vivo comet studies)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MAIN EXPERIMENT 1 RESULTS
Formulations Analysis
The formulations analysis results are presented in the Formulation Analysis Phase Report.
Results of the analyses demonstrated that the formulations were homogeneous with a relative standard deviation (RSD) =5%.

Achieved concentration values were 87.5, 87.4 and 78.8% nominal at 33.33, 66.67 and 133.33 mg/mL and therefore fell below protocol specification criteria of 90-100% nominal. Therefore, the animals received doses of 437, 875 and 1575 mg/kg/day. It was therefore decided to report the actual doses achieved and perform some additional dosing at 133.33 mg/mL to achieve the regulatory maximum dose level of 2000 mg/kg/day.

For group 1, a small peak was detected on the vehicle chromatogram at the analyte retention time. As its area was below the LOQ area, this peak was considered as negligible as its concentration could be estimated to be below 0.1 mg/mL.

Post Dose Observations
No clinical signs of toxicity were observed in any animal following treatments with vehicle, Diethylaminopropylamine (at 437, 875 or 1575 mg/kg/day) or the positive control (EMS).

Body Weights
No notable effect of treatment on body weights was observed.

Clinical Pathology
Diethylaminopropylamine-related clinical pathology changes consisted of minimal decreases in potassium and minimal increases in chloride in all groups administered Diethylaminopropylamine. A minimal increase in phosphate was recorded for animals administered 875 or 1575 mg/kg/day. Minimal increases in creatinine and/or urea were recorded for animals administered =437 mg/kg/day.

Histopathology
Macroscopic and Microscopic findings are presented in the Pathology Contributory Report.
On macroscopic examination, there were no changes which were considered related to Diethylaminopropylamine.

On microscopic examination, Diethylaminopropylamine-related microscopic observations were noted in the stomach and duodenum. In the stomach, edema at the limiting ridge and vacuolation of the superficial mucosa were recorded for animals administered 875 or 1575 mg/kg/day. In the liver, decreased hepatocyte glycogen was recorded for animals administered 1575 mg/kg/day.

MAIN EXPERIMENT 2 RESULTS
Formulations Analysis
The formulations analysis results are presented in the Formulation Analysis Phase Report.
Results of the analyses demonstrated that the formulations were homogeneous with a relative standard deviation (RSD) =5%.

The achieved concentration value was 96.5% nominal at 133.33 mg/mL and therefore met protocol specification criteria of 90-100% nominal.

No test article was detected in the vehicle sample.

Overall, the data were considered acceptable.

Post Dose Observations
No clinical signs of toxicity were observed in any animal following treatments with vehicle, Diethylaminopropylamine (at 2000 mg/kg/day) or the positive control (EMS).

Body Weights
No notable effect of treatment on body weight at 2000 mg/kg/day was observed.

Clinical Pathology
Diethylaminopropylamine-related clinical pathology changes consisted of minimal decreases in potassium and minimal increases in chloride, sodium and phosphate. Minimal increases in creatinine and/or urea were recorded.

Histopathology
Macroscopic and Microscopic findings are presented in the Pathology Contributory Report.
On macroscopic examination, there were no changes which were considered related to Diethylaminopropylamine.

On microscopic examination, Diethylaminopropylamine-related microscopic observations were noted in the stomach, duodenum and liver. In the stomach, edema at the limiting ridge and vacuolation of the superficial mucosa were recorded for animals administered 2000 mg/kg/day. In the duodenum, vacuolation of mucosal epithelial cells and/or atrophy of the villi were recorded for animals in administered 2000 mg/kg/day. In the liver, decreased hepatocyte glycogen was recorded for animals administered 2000 mg/kg/day.

Any other information on results incl. tables

Range-finder Results

In the Range-Finder Experiment, groups of 3 male and/or 3 female rats were dosed with Diethylaminopropylamine at either 2000 (males and females) or 1400 mg/kg/day (females only).

At 2000 mg/kg/day in male animals following the first dose, clinical signs of piloerection and decreased activity were observed at 2 and 4-6 hours. All males were back to a normal state prior to dosing on Day 2 and no further clinical signs were seen. At 2000 mg/kg/day in female animals following the first dose, clinical signs of toxicity were observed 2/3 animals and included piloerection and/or decreased activity. Prior to dosing on Day 2, a single female animal was found dead during the morning health checks. In the remaining females following the second dose, one animal displayed transient mouth rubbing immediately following dosing. The other female started to display clinical signs from 0.5 hours post-dose which became more severe with time to the end of the observation period. These included decreased activity (from 0.5 hours), hunched posture, ptosis (from 2 hours), piloerection, anogenital soiling, ptosis and staining around the snout (at approximately 4 hours). No losses in bodyweight were seen in the surviving animals. Necropsy of the decedent identified no obvious cause of death.

At 1400 mg/kg/day in females, the dose was well tolerated with no clinical signs of toxicity observed following either dose and no notable losses in animal bodyweight.

As there was no substantial difference in dose tolerability between the sexes, male animals only were used in the Main Experiment.

Based on these data the regulatory maximum dose of 2000 mg/kg/day was considered a suitable high dose to be used in the Main Experiment with intermediate and low doses of 1000 and 500 mg/kg/day, respectively.

 

Validity of Data

For Experiments 1 and 2, the data generated in this study confirm that:

1. The vehicle control data fell within the laboratory’s historical vehicle control data for each tissue


2. The positive control induced responses that were comparable with the laboratory’s historical positive control data and produced a statistically significant increase in TI compared to the concurrent vehicle control for each tissue


3. Adequate numbers of cells and doses were analysed


4. The high dose in Main Experiment 2 was considered to be the regulatory maximum recommended dose.

The Study Monitor confirmed no bioanalysis was required on this study as it was considered that there was enough information generated in the 90 day toxicity study (CiToxLAB Study Number 42760) to suggest exposure.

In regards to confirmation of target tissue exposure confirmed in this study, the dose route selected (oral gavage) ensured that site of contact tissues (stomach and duodenum) were exposed. In addition, microscopic observations in the stomach (edema at the limiting ridge and vacuolation of the superficial mucosa recorded for animals administered 875, 1575 or 2000 mg/kg/day) and the duodenum (vacuolation of mucosal epithelial cells and/or atrophy of the villi) for animals administered 2000 mg/kg/day, are suggestive of tissue toxicity or adaptive changes further confirming exposure to Diethylaminopropylamine.

The assay data were therefore considered valid.

Data Analysis

For both Main Experiments 1 and 2, there were no marked, dose related increases in hedgehogs in liver, stomach or duodenum thus demonstrating that treatment with Diethylaminopropylamine did not cause excessive DNA damage that could have interfered with comet analysis (Text Table 1 to Text Table 6).

Experiment 1
In the liver and stomach, male animals treated with Diethylaminopropylamine at all doses displayed group mean (Text Table 1 and Text Table 2) and individual animal TI values which were similar to the concurrent vehicle control group and fell within the laboratory’s historical vehicle control 95% reference range. There were no statistically significant increases in TI in liver or stomach for any of the groups receiving the test article, compared to the concurrent vehicle control group and no evidence of a dose-response, indicating a negative result in both tissues up to 1575 mg/kg/day.

In the duodenum, male animals treated with Diethylaminopropylamine at the intermediate dose level of 875 mg/kg/day demonstrated a statistically significant (P≤0.05) increase in group mean TI of 1.90 % which contributed to a statistically significant linear trend (Text Table 3.). However, the increase was primarily due to a single animal (R0202) within the group demonstrating an elevated TI value of 6.98 %. Although there were no corresponding pathology findings to suggest target tissue toxicity, the increases were concomitant with a marked increase in %hedgehogs (highly damaged cells) in this animal, to 17.84% (historical vehicle control observed range of 0 to 17.39 % hedgehogs). Therefore, it was likely that the increase in TI in this animal was due to mechanical damage during tissue processing rather than a true genotoxic effect and given this was isolated to a single animal, it was considered the statistically significant increase in TI was not biologically relevant.

Experiment 2
In the liver and stomach, male animals treated with Diethylaminopropylamine at 2000 mg/kg/day displayed group mean (Text Table 4 and Text Table 5) and individual animal TI values that were similar to the concurrent vehicle control group and fell within the laboratory’s historical vehicle control ranges . There were no statistically significant increases in TI in liver or stomach at 2000 mg/kg/day, compared to the concurrent vehicle control group, indicating a clear negative result in both tissues at 2000 mg/kg/day.

In the duodenum, male animals treated with Diethylaminopropylamine at 2000 mg/kg/day demonstrated a small, but statistically significant (P≤0.05) increase in group mean TI to 1.42 % (Text Table 6). However, the increase was primarily due to a single animal (R0602) within the group demonstrating an elevated TI value of 5.31 %. There were also corresponding pathology findings which were suggestive of target tissue toxicity (atrophy of the villi). Therefore, as the individual animal and group mean TI values fell within the laboratory’s historical vehicle 95% reference range and the increase was isolated to a single animal and concomitant with histopathological changes, it was considered the statistically significant increase in TI was not biologically relevant.

 

Text Table 1: Diethylaminopropylamine: Summary of Group Mean Data – Experiment 1, Liver

Group/ Dose Level	Tail Intensity						Mean % Hedgehogs
(mg/kg/day)	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
1/ Vehicle (0)	0.22	0.12	-	-	-	-	0.88
2/ Diethylaminopropylamine (437)*	0.33	0.11	2.39	U	0.2831	NS	1.61
3/ Diethylaminopropylamine (875)*	0.09	0.03	0.30	U	0.9905	NS	2.06
4/ Diethylaminopropylamine (1575)*	0.29	0.06	2.34	U	0.2939	NS	1.45
5/ EMS (200)	13.33	2.16	183.64	U	0.0002	P≤0.001	0.55
Dose response: (groups 1,2,3,4)				U	0.4234	NS	N/A

* Due to lower than expected analytical results, the actual dose levels administered have been reported.
SEM	Standard Error of Mean
NS	Not significant (P>0.05)
U	Unranked

 

Text Table 2: Diethylaminopropylamine: Summary of Group Mean Data – Experiment 1, Stomach

Group/ Dose Level	Tail Intensity						Mean % Hedgehogs
(mg/kg/day)	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
1/ Vehicle (0)	0.31	0.05	-	-	-	-	7.77
2/ Diethylaminopropylamine (437)*	0.29	0.05	0.87	U	0.8885	NS	9.35
3/ Diethylaminopropylamine (875)*	0.21	0.03	0.75	U	0.9577	NS	6.66
4/ Diethylaminopropylamine (1575)*	0.24	0.05	0.77	U	0.9508	NS	7.00
5/ EMS (200)	13.72	1.86	58.59	U	<0.0001	P≤0.001	7.91
Dose response: (groups 1,2,3,4)				U	0.8302	NS	N/A

 

 

* Due to lower than expected analytical results, the actual dose levels administered have been reported.
SEM	Standard Error of Mean
NS	Not significant (P>0.05)
U	Unranked

 

Text Table 3: Diethylaminopropylamine: Summary of Group Mean Data – Experiment 1, Duodenum

Group/ Dose Level	Tail Intensity						Mean % Hedgehogs
(mg/kg/day)	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
1/ Vehicle (0)	0.47	0.13	-	-	-	-	9.92
2/ Diethylaminopropylamine (437)*	0.46	0.15	1.08	U	0.6858	NS	6.88
3/ Diethylaminopropylamine (875)*	1.90	1.05	3.10	U	0.0353	P≤0.05	6.84
4/ Diethylaminopropylamine (1575)*	0.72	0.18	1.88	U	0.2165	NS	5.01
5/ EMS (200)	5.52	0.41	16.59	U	0.0005	P≤0.001	9.24
Dose response: (groups 1,2,3,4 )				U	0.0326	P≤0.05	N/A

 

 

* Due to lower than expected analytical results, the actual dose levels administered have been reported.
SEM	Standard Error of Mean
NS	Not significant (P>0.05)
U	Unranked

 

Text Table 4: Diethylaminopropylamine: Summary of Group Mean Data – Experiment 2, Liver

Group/ Dose Level	Tail Intensity						Mean % Hedgehogs
(mg/kg/day)	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
6/ Vehicle (0)	0.08	0.02	-	-	-	-	1.15
7/ Diethylaminopropylamine (2000)	0.10	0.02	1.26	U	0.1725	NS	1.79
8/ EMS (200)	16.64	1.80	237.47	U	<0.0001	P≤0.001	2.63
Dose response not performed				N/A	N/A	N/A	N/A

 

SEM	Standard Error of Mean
NS	Not significant (P>0.05)
U	Unranked

 

Text Table 5: Diethylaminopropylamine: Summary of Group Mean Data – Experiment 2, Stomach

Group/ Dose Level	Tail Intensity						Mean % Hedgehogs
(mg/kg/day)	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
6/ Vehicle (0)	0.68	0.18	-	-	-	-		3.69
7/ Diethylaminopropylamine (2000)	0.49	0.10	0.74	U	0.7988	NS		5.25
8/ EMS (200)	11.81	1.37	22.13	U	0.0001	P≤0.001		5.03
Dose response not performed				N/A	N/A	N/A		N/A

 

SEM	Standard Error of Mean
NS	Not significant (P>0.05)
U	Unranked

 

Text Table 6: Diethylaminopropylamine: Summary of Group Mean Data – Experiment 2, Duodenum

Group/ Dose Level	Tail Intensity						Mean % Hedgehogs
(mg/kg/day)	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
6/ Vehicle (0)	0.32	0.08	-	-	-	-	7.32
7/ Diethylaminopropylamine (2000)	1.42	0.79	3.48	U	0.0372	P≤0.05	6.19
8/ EMS (200)	5.32	0.70	26.43	U	0.0005	P≤0.001	6.96
Dose response not performed				N/A	N/A	N/A	N/A

 

SEM	Standard Error of Mean
NS	Not significant (P>0.05)
U	Unranked

 

 

Applicant's summary and conclusion

Conclusions:

It is concluded that, under the conditions of this comet assay, Diethylaminopropylamine did not induce biologically relevant increases in DNA strand breaks in the liver, stomach or duodenum when tested up to 2000 mg/kg/day (the maximum recommended dose for in vivo comet studies).

Executive summary:

Introduction

The in vivo alkaline comet  - OECD Guideline 489, (single cell gel electrophoresis) assay is used for the detection of DNA strand breaks (apparent as an increase in DNA migration) in cells or nuclei isolated from tissues of animals that have been exposed to potentially genotoxic material(s). Under alkaline conditions (>pH 13), the comet assay can detect single and double stranded breaks, resulting, for example, from direct interactions with DNA, alkali labile sites or as a consequence of transient DNA strand breaks resulting from DNA excision repair. These strand breaks may be repaired, resulting in no persistent effect, may be lethal to the cell, or may be fixed into a mutation resulting in a permanent viable change. They may also lead to chromosomal damage which is also associated with many human diseases including cancer.

Clinical signs of toxicity:	Experiment 1 and Experiment 2: None.
Tissues sampled:	Liver, stomach, duodenum and gonad were sampled on Day 2, equivalent to 24 hours.
Formulation analysis:	Experiment 1: Achieved concentration values for all formulations fell below protocol specification criteria of 90-100% nominal. Homogeneity of all formulations were within protocol specification (%RSD ≤5%). A small peak was detected on the vehicle control (Group 1) chromatogram at the analyte time. As its area was below the LOQ area, this peak was considered as negligible (below 0.1 mg/mL). Experiment 2: Achieved concentration met protocol specification criteria of 90-100% nominal. Homogeneity was within protocol specification (%RSD ≤5%). No test article was detected in the vehicle control sample.
Clinical Chemistry:	Diethylaminopropylamine-related clinical pathology changes consisted of minimal decreases in potassium and minimal increases in chloride in all groups administered Diethylaminopropylamine and minimal increases in sodium in animals administered 2000 mg/kg/day. A minimal increase in phosphate was recorded for animals administered 875, 1575 or 2000 mg/kg/day. Minimal increases in creatinine and/or urea were recorded for animals administered ≥437 mg/kg/day.
Histopathology:	On macroscopic examination, there were no changes which were considered related to Diethylaminopropylamine. On microscopic examination, Diethylaminopropylamine-related microscopic observations were noted in the stomach, duodenum and liver. In the stomach, edema at the limiting ridge and vacuolation of the superficial mucosa were recorded for animals administered 875, 1575 or 2000 mg/kg/day. In the duodenum, vacuolation of mucosal epithelial cells and/or atrophy of the villi were recorded for animals in administered 2000 mg/kg/day. In the liver, decreased hepatocyte glycogen was recorded for animals administered 1575 or 2000 mg/kg/day.
Assay validity (Experiments 1 and 2):	For liver, stomach and duodenum, the vehicle control %tail intensity (TI) data fell within the laboratory’s historical vehicle control ranges. For liver, stomach and duodenum, the positive control induced a statistically significant increase in TI (over the current vehicle control group) that fell within the laboratory’s historical positive control ranges. The assay was therefore accepted as valid.

 

For both Main Experiments 1 and 2, there were no marked, dose related increases in hedgehogs in liver, stomach or duodenum thus demonstrating that treatment with Diethylaminopropylamine did not cause excessive DNA damage that could have interfered with comet analysis.

Experiment 1
In the liver and stomach, male animals treated with Diethylaminopropylamine at all doses displayed group mean and individual animal TI values which were similar to the concurrent vehicle control group and fell within the laboratory’s historical vehicle control 95% reference range. There were no statistically significant increases in TI in liver or stomach for any of the groups receiving the test article, compared to the concurrent vehicle control group and no evidence of a dose-response, indicating a negative result in both tissues up to 1575 mg/kg/day.

In the duodenum, male animals treated with Diethylaminopropylamine at the intermediate dose level of 875 mg/kg/day demonstrated a statistically significant (P≤0.05) increase in group mean TI of 1.90% which contributed to a statistically significant linear trend. However, the increase was primarily due to a single animal (R0202) within the group demonstrating an elevated TI value of 6.98%. Although there were no corresponding pathology findings to suggest target tissue toxicity, the increases were concomitant with a marked increase in %hedgehogs (highly damaged cells) in this animal, to 17.84% (historical vehicle control observed range of 0 to 17.39% hedgehogs). Therefore, it was likely that the increase in tail intensity in this animal was due to mechanical damage during tissue processing rather than a true genotoxic effect and given this was isolated to a single animal, it was considered the statistically significant increase in TI was not biologically relevant.

Experiment 2
In the liver and stomach, male animals treated with Diethylaminopropylamine at 2000 mg/kg/day displayed group mean and individual animal TI values which were similar to the concurrent vehicle control group and fell within the laboratory’s historical vehicle control ranges. There were no statistically significant increases in TI in liver or stomach at 2000 mg/kg/day, compared to the concurrent vehicle control group, indicating a clear negative result in both tissues at 2000 mg/kg/day.

In the duodenum, male animals treated with Diethylaminopropylamine at 2000 mg/kg/day demonstrated a small, but statistically significant (P≤0.05) increase in group mean TI to 1.42 %. However, the increase was primarily due to a single animal (R0602) within the group demonstrating an elevated TI value of 5.31 %. There were also corresponding pathology findings which were suggestive of target tissue toxicity (atrophy of the villi). Therefore, as the individual animal and group mean TI values fell within the laboratory’s historical vehicle 95% reference range and the increase was isolated to a single animal and concomitant with histopathological changes, it was considered the statistically significant increase in TI was not biologically relevant.

It is concluded that, under the conditions of this comet assay, Diethylaminopropylamine did not induce biologically relevant increases in DNA strand breaks in the liver, stomach or duodenum when tested up to 2000 mg/kg/day (the maximum recommended dose for in vivo comet studies).