Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with diethylamine

Genetic toxicity: in vivo

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:

Experimental Start Date: 07 March 2022
Experimental Completion Date: 28 April 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)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with diethylamine

EC Number:

500-425-6

EC Name:

Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with diethylamine

Cas Number:

159034-91-0

Molecular formula:

Not available for this UVCB

IUPAC Name:

Reaction product of poly(oxy-1,2-ethanediyl), .alpha.-hydro-.omega.-[(1-oxo-2-propenyl)oxy]-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1) and N-ethylethanamine

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Justification for Selection
The comet assay is recommended by various regulatory authorities as an appropriate test to determine the genotoxic potential of a compound in vivo (OECD, 2016). This study has been designed to provide data at the request of the European chemical agency (ECHA), based on Article 40 of the REACH regulation (Regulation (EC) No 1907/2006).

The rat was selected as there is a large volume of background data in this species and has been specifically requested by the authorities.

The liver is recommended as the primary site of xenobiotic metabolism and is often highly exposed to both the test article and any metabolites. The stomach and duodenum are recommended tissues to examine for site of contact effects after oral exposure. Male gonadal cells will be collected for potential analysis if induction of DNA strand breaks is determined in any somatic tissue. The analysis of gonadal cells may be relevant for the overall assessment of possible germ cell mutagenicity including classification and labelling according to CLP Regulation. As such, this study will be performed in male rats only.

Sex:

male

Details on test animals or test system and environmental conditions:

Species, Strain and Supplier
33 male young adult out-bred Sprague Dawley rats (HSD:Sprague DawleySD) were obtained from Envigo, Blackthorn, UK.

Animals not dosed in this study were transferred to Labcorp Early Development Laboratories Ltd. stock.

Specification
3 animals were dosed during the Range-Finder Experiment. They were approximately 7 to 8 weeks old and 194-221 g on the first day of dosing.

27 animals were dosed during the Main Experiment. They were approximately 7 to 8 weeks old and 194-235 g on the first day of dosing.


Environment
Animals were housed in wire topped, solid bottomed cages, with three animals per cage.
The animals were housed in rooms air-conditioned to provide a minimum of 15 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 Labcorp Early Development Laboratories Ltd.

Allocation to Treatment Group
On arrival, animals were randomly allocated to cages. Range-Finder animals 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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

The vehicle was selected as it has been used in previous in vivo studies with this compound.

Details on exposure:

Test Article Formulation
Preparation
Formulations were freshly prepared prior to each dosing occasion for the Range-Finder and prepared once for each dose group in the Main Experiment. Acrylated amine synergist (CN3715) was formulated in corn oil as follows:

The test article was weighed out into a pre-labelled bottle and vehicle was added to achieve the final volume. Formulations were stirred on a magnetic stirrer to homogenise and aliquoted (as required).

Stability
No stability information was provided by the Sponsor prior to the start of the study. Consequently, formulations for the Range-Finder were stored at 15-25°C, protected from light, and used within 2 hours of preparation. The formulations were assumed stable for this period as a non-GLP study determined test article formulations at 5 and 200 mg/mL were stable when stored for 4 days at +4°C, protected from light.

Stability data generated in this study prior to the Main Experiment confirmed formulations of Acrylated amine synergist (CN3715) in corn oil at 50 and 200 mg/mL were stable for 3 days when stored at 15-25°C, protected from light.

Consequently, all test article formulations for the Main Experiment were stored at 15-25°C, protected from light, and used within 24 hours of preparation.

Homogeneity
To ensure homogeneity, dose formulations were stirred continuously (on a magnetic stirrer) before and throughout dosing.

Formulations Analysis
Samples were taken from each article formulation used in the Main Experiment together with concurrent vehicle controls.

Duplicate (2 x 1 mL) samples were taken from the top, middle and bottom of each test article formulation together with a single 1 mL sample (taken from the middle) of the vehicle control and all samples were analysed for achieved concentration and a determination of homogeneity.

Samples were stored at 15-25°C, prior to analysis by Labcorp Early Development Laboratories Ltd.
The analytical method is detailed in the Formulation Analysis Contributory Report.

Stability Assessment
Test article formulations at 50 and 200 mg/mL were assessed for stability.

Duplicate (2 x 1 mL) samples were taken from the top, middle and bottom of each test article formulation and all samples were analysed for achieved concentration and a determination of homogeneity.

The remaining bulk formulation was split into 2 aliquots; one aliquot was stored at room temperature (15-25°C) and triplicate samples were taken and analysed for achieved concentration on Day 1 and Day 3 and the remaining aliquot was stored at 2 8ºC. As stability was confirmed at room temperature no sampling or analysis of the refrigerated aliquot was performed.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

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

Main Experiment animals were dosed in replicate cage order i.e. cage 1 of Groups 1-4 dosed in ascending group order then cage 2 of Groups 1-4 in ascending group order. Group 5 was dosed at a time that allowed necropsy of these animals after Group 4 necropsy. Animals were not fasted prior to administration and were sampled at 24 hours.

Post exposure period:

Observation times were as follows:

Animals Day Approximate Observation Time
Range-Finder Experiment 1 Prior to dose, immediate, 0.5, 1, 2 and 4-6 hours post dose
2 Prior to dose, immediate, 0.5, 1, 2 and 4-6 hours post dose

Main Experiment 1* Prior to dose, immediate, 1, 2 and 4 hours post dose
2 Prior to dose, immediate and prior to necropsy
* Excluding positive control group

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Range-finder experiment

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 1 - Vehicle control

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 5

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Group 4

Doses / concentrations 6

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Group 5 - Positive cntrol

No. of animals per sex per dose:

6 males per dose group, including vehicle control
3 males - positive control

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:

Liver, stomach, duodenum, gonad*
* Left testis for Comet; Right for Histopathology

The liver, stomach, duodenum and gonad were removed from each control (vehicle and positive) and test article treated animal.
For histopathology, a sample of liver, stomach, duodenum and gonad from vehicle control and test article treated animals only was removed. Liver, stomach and duodenum samples were immediately preserved in neutral buffered formalin and stored at room temperature. Gonad samples were immediately preserved in modified Davidson’s fluid and stored at room temperature. No histopathology samples were preserved for the positive control animals.

Details of tissue and slide preparation:

Justification for Dose Selection
The following information on the in vivo toxicity of Acrylated amine synergist (CN3715) was provided:

In an acute toxicity study, CN3715 was administered once by oral route (gavage) to two groups of three fasted female Sprague-Dawley rats at the dose-level of 2000 mg/kg (in corn oil). No unscheduled deaths occurred during the study. Piloerection was observed in 4/6 females within 4 hours after treatment. No clinical signs persisted from day 2. The test item administration did not induce any macroscopic changes.

A preliminary study was performed to evaluate the potential toxicity of the test article, Acrylated amine synergist (CN3715), following daily oral administration (5 mL/kg via gavage) to Sprague Dawley rats for 2 weeks. Groups of three male and three female rats were dosed at 100, 300 or 1000 mg/kg/day. There were no unscheduled deaths and clinical observations were limited to hypersalivation. All dose levels were well tolerated with a maximum tolerated dose considered to exceed 1000 mg/kg/day in males and females.

A Range-Finder Experiment was performed in this study to confirm that there is no impact on dose tolerability for unfasted male animals (compared to fasted female animals in the previous acute toxicity study) under the dosing regimen of this study (two administrations). An initial dose of 2000 mg/kg/day (the regulatory recommended maximum dose) was administered in the Range-Finder Experiment (OECD, 2016).

From the results of the Range-Finder Experiment dose levels of 500, 1000 and 2000 mg/kg Acrylated amine synergist (CN3715) (equivalent to 25%, 50% of the maximum dose and the maximum dose respectively) were tested in the Main Experiment.

Histopathology
Preserved liver, stomach, duodenum and gonad samples were embedded in wax blocks and sectioned at 5 µm nominal. Liver, stomach and duodenum slides were stained with haematoxylin and eosin and examined by the Study Pathologist. Gonad samples were not examined as the somatic tissues did not show genotoxic potential.

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 ice cold Merchants solution to produce single cell suspensions.

The comet stomach samples were washed in ice cold Merchants solution and then incubated on ice for 15 minutes prior to processing. After incubation the stomach samples were removed from the Merchants solution and the inner surface gently scraped twice (released material discarded) using the back of a scalpel blade. Cells were gently scraped from the inside surface of the stomach using the back of a scalpel blade in 200 µL of fresh Merchants solution to produce single cell suspensions.

The comet duodenum samples were washed thoroughly in ice cold Merchants solution; each sample was vortexed in ice cold Merchants solution for approximately 15 seconds. The tissue was removed from the Merchants solution and the inner surface gently scraped twice (released material discarded) using the back of a scalpel blade. The tissue was vortexed in ice cold Merchants solution for a further 15 seconds prior to gently scraping the inside of the duodenum three times with the back of a scalpel blade in 150 µL of fresh Merchants solution to produce single cell suspensions.

The comet gonad samples were prepared by making an incision along the length of a single gonad, removing the contents from the membrane and discarding the membrane. The remaining tissue was cut into small pieces and gently 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 by an individual not connected to scoring of the study.

All 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.

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:

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 both of the above criteria were met.
The test article was considered negative in this assay if neither 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, consistency of response within and between dose levels and any confirmatory experiments.

Statistics:

Please refer to "Any other information on materials and methods"

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDER RESULTS
A group of three male rats was dosed at 0 hours and approximately 21 hours with Acrylated amine synergist (CN3715) at 2000 mg/kg/day. No clinical signs of toxicity were observed for any animal for the duration of the observation period. All 3 males increased in body weight between Day 1 and Day 3.

From these results the regulatory limit dose of 2000 mg/kg/day was tolerated. This was selected as the maximum dose for the Main Experiment. Two lower doses of 500 and 1000 mg/kg/day (equivalent to 25% and 50% of the maximum dose) were also selected.


Post Dose Observations
.There were no clinical observations of toxicity for any animal dosed with the vehicle, Acrylated amine synergist (CN3715) (at 500, 1000 or 2000 mg/kg/day) or the positive control (EMS).

Body Weights
There was no clear test article related impact on animal bodyweights between Day 1 and Day 2 with group mean body weight change values of +1.0%, +2.1% and +2.0% for 500, 1000 and 2000 mg/kg/day, respectively, compared to +1.1% for the concurrent vehicle control group.

Bioanalysis
Plasma was processed from whole blood samples as a contingency for systemic exposure confirmation. Analysis of these samples was not performed.

Clinical Pathology
Clinical Chemistry

There were no Acrylated amine synergist (CN3715)-related clinical chemistry changes recorded. Any differences in individual clinical pathology parameters observed for animals administered the test article were considered not test article related due to the negligible magnitude of the change, individual animal variability, and overlap of values for test article treated animals with concurrent control values.

Histopathology
No macroscopic or microscopic changes were considered related to Acrylated amine synergist (CN3715).

Examined tissues were considered macroscopically or microscopically unremarkable, or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

Data Analysis
There were no dose-related increases in %hedgehogs in liver, stomach or duodenum, thus demonstrating that treatment with Acrylated amine synergist (CN3715) did not cause excessive DNA damage that could have interfered with comet analysis.

Animals treated with Acrylated amine synergist (CN3715) at all doses exhibited group mean tail intensities in the liver that fell within the 95% reference range of the laboratory’s historical vehicle control data. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, and the statistical assessment for dose response was not significant (p > 0.05).

There were two individual animal liver %tail intensity values at 2000 mg/kg/day (R0301, 2.39% and R0303, 3.52%) that exceeded the upper limit of the 95% reference range (1.80%). However, these increases above the concurrent vehicle control group and the 95% reference range were not reproduced in all animals at 2000 mg/kg/day, did not increase the group mean above the 95% reference range and did not contribute to a statistically significant increase for the group compared to the vehicle control group. None of the evaluation criteria for a positive result were met and therefore, these isolated data were considered of no biological relevance.

Animals treated with Acrylated amine synergist (CN3715) at all doses exhibited group mean tail intensities in the stomach that fell within the laboratory's the 95% reference range of the laboratory’s historical vehicle control data. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control. There was a statistically significant dose response noted, however, this response was driven predominantly by an isolated animal response at 2000 mg/kg/day (R0301). All individual animal stomach %tail intensity values fell within the 95% reference range, however the %tail intensity for animal R0301 (5.31%) was elevated compared to all other vehicle and test article dosed animals (0.08-1.01%). As it was within the 95% reference range, it fell within the normal variation of the assay and was likely induced by the mechanical processing of the tissue.
Overall, it was considered that this isolated animal value, which increased the group mean and contributed to the statistically significant dose response, but was not reproduced in other animals and did not contribute to a statistically significant increase in tail intensity at 2000 mg/kg/day compared to the vehicle control group, was of no biological relevance.

Animals treated with Acrylated amine synergist (CN3715) at all doses exhibited group mean tail intensities in the duodenum that fell within, or marginally below, the 95% reference range of the laboratory’s historical vehicle control data. There was a statistically significant increase in tail intensity at the intermediate dose (1000 mg/kg/day) group only, compared to the concurrent vehicle control; both the low dose (500 mg/kg/day) and high dose (2000 mg/kg/day) groups were not statistically significantly increased compared to the concurrent vehicle control. As all individual animal and group mean %tail intensity values fell within, or marginally below, the 95% reference range (moreover all values fell below the mean value of the laboratory’s historical vehicle control data indicate value fell towards the lower end of normal variation within the assay), and there was no evidence of a dose response, the statistical significance associated with the intermediate dose group was considered of no biological relevance.

These data were considered negative in the liver, stomach and duodenum. Therefore, no analysis of gonad slides is required.

Any other information on results incl. tables

Formulations Analysis

Analyses demonstrated that the test article formulations at 50, 100 and 200 mg/mL were homogeneous (0.67-1.17% relative standard deviation (RSD), which fell within target criteria of ≤5%) and met criteria (100±15% of the nominal test article concentrations) for acceptable achieved concentration (mean values of 99-100%). The formulations were therefore considered acceptable. No test article was detected in the vehicle sample.

 

Validity of Data

The data generated in this study confirm that:

1. The vehicle control data were comparable to laboratory historical control data for each tissue


2. The positive control induced responses that were compatible with the laboratory historical control data and produces are statistically significant compared to the concurrent vehicle control


3. Adequate numbers of cells and doses were analysed


4. The high dose was considered to be the maximum recommended dose.

As dosing was via oral gavage, exposure to the stomach and duodenum was assured. With regards to the liver, there were no clinical observations of toxicity and no clinical chemistry changes or histopathological changes related to the test article to provide indirect evidence of systemic exposure. Publically available data suggest Acrylated amine synergist (CN3715) is a UVCB whose constituents have a molecular weight ranging from 369 to 820 g/mol. At 20°C, the substance is soluble in water at ca. 59 g/L and has a octanol:water partition coefficient (Log Kow) ranging from 1.05 to 4.08 (GRL, 2012 and GRL, 2013). According to ECHA (2017), these properties are favourable for absorption following exposure via the oral route. Therefore, while this study did not show effects that could only be attributed to indirect systemic exposure to the test article and/or its metabolites (exposure does not have to be confirmed concurrently within the study, OECD, 2016), absorption is considered very likely by the sponsor based on the physicochemical properties of Acrylated amine synergist (CN3715).

The assay data were therefore considered valid.

 

Text Table 1: Acrylated amine synergist (CN3715): Summary of Group Mean Data – Liver

Group/Dose Level (mg/kg/day)	Tail Intensity						Mean % Hedgehogs
	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
1/ Vehicle (0)	0.32	0.07	-	-	-	-	0.75
2/ Acrylated amine synergist (CN3715) (500)	0.42	0.09	1.08	R	0.7354	NS	0.62
3/ Acrylated amine synergist (CN3715) (1000)	0.22	0.06	0.59	R	0.9794	NS	1.07
4/ Acrylated amine synergist (CN3715) (2000)	1.31	0.59	1.69	R	0.8279	NS	1.01
5/ EMS (200)	19.16	0.65	67.56	U	<0.0001	***	1.20
Dose response: (groups 1,2,3,4 )				R	0.7333	NS	N/A

 

Text Table 2: Acrylated amine synergist (CN3715): Summary of Group Mean Data – Stomach

Group/Dose Level (mg/kg/day)	Tail Intensity						Mean % Hedgehogs
	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
1/ Vehicle (0)	0.22	0.05	-	-	-	-	5.25
2/ Acrylated amine synergist (CN3715) (500)	0.39	0.07	1.55	U	0.3765	NS	7.73
3/ Acrylated amine synergist (CN3715) (1000)	0.59	0.12	2.48	U	0.0946	NS	6.74
4/ Acrylated amine synergist (CN3715) (2000)	1.21	0.83	2.37	U	0.1117	NS	6.89
5/ EMS (200)	12.91	4.04	67.17	U	<0.0001	***	6.50
Dose response: (groups 1,2,3,4 )				U	0.0319	*	N/A

 

EMS	Ethyl Methanesulfonate
SEM	Standard Error of Mean
N/A	Not applicable
NS	Not significant (P>0.05)
*	P≤0.05
***	P≤0.001
R	Ranked
U	Unranked

 

Text Table 3: Acrylated amine synergist (CN3715): Summary of Group Mean Data – Duodenum

Group/Dose Level (mg/kg/day)	Tail Intensity						Mean % Hedgehogs
	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
1/ Vehicle (0)	0.15	0.02	-	-	-	-	9.65
2/ Acrylated amine synergist (CN3715) (500)	0.20	0.03	1.38	U	0.2016	NS	11.42
3/ Acrylated amine synergist (CN3715) (1000)	0.38	0.05	2.62	U	0.0007	***	10.23
4/ Acrylated amine synergist (CN3715) (2000)	0.15	0.03	1.01	U	0.7271	NS	9.86
5/ EMS (200)	6.01	0.37	54.30	U	<0.0001	***	14.34
Dose response: (groups 1,2,3,4 )				U	0.1800	NS	N/A

 

EMS	Ethyl Methanesulfonate
SEM	Standard Error of Mean
N/A	Not applicable
NS	Not significant (P>0.05)
***	P≤0.001
U	Unranked

 

Table 9.5: Liver: Animal Comet Data

Group/ Dose Level	Animal		Total	Tail Intensity (%)		Hedgehogs
(mg/kg/day)	Number		Comets	Mean	SD	(%)
1/ Vehicle (0)	R0001		150	0.62	0.23	0.57
	R0002		150	0.20	0.07	0.57
	R0003		150	0.39	0.03	1.71
	R0004		150	0.21	0.04	0.00
	R0005		150	0.35	0.16	0.55
	R0006		150	0.17	0.04	1.01
2/ Acrylated amine synergist (CN3715) (500)	R0101		150	0.33	0.19	1.69
	R0102		150	0.17	0.17	0.00
	R0103		150	0.77	0.24	1.18
	R0104		150	0.56	0.30	0.48
	R0105		150	0.46	0.22	0.00
	R0106		150	0.23	0.22	0.48
3/ Acrylated amine synergist (CN3715) (1000)	R0201		150	0.49	0.15	1.15
	R0202		150	0.11	0.11	2.91
	R0203		150	0.23	0.20	0.54
	R0204		150	0.12	0.04	0.49
	R0205		150	0.23	0.20	0.52
	R0206		150	0.14	0.05	1.01
4/ Acrylated amine synergist (CN3715) (2000)	R0301		150	2.39	0.97	0.55
	R0302		150	0.15	0.13	0.63
	R0303		150	3.52	0.67	0.59
	R0304		150	0.10	0.04	3.00
	R0305		150	1.62	0.45	0.00
	R0306		150	0.08	0.03	1.01
5/ EMS (200)	R0401		150	18.02	5.05	2.11
	R0402		150	19.18	1.47	0.48
	R0403		150	20.28	1.57	1.09
SD		Standard Deviation
EMS		Ethyl Methanesulfonate

 

 

Table 9.7: Stomach: Animal Comet Data

Group/ Dose Level	Animal		Total	Tail Intensity (%)		Hedgehogs
(mg/kg/day)	Number		Comets	Mean	SD	(%)
1/ Vehicle (0)	R0001		150	0.37	0.28	8.47
	R0002		150	0.36	0.10	1.72
	R0003		150	0.08	0.04	2.47
	R0004		150	0.19	0.16	5.92
	R0005		150	0.14	0.10	6.13
	R0006		150	0.19	0.11	6.56
2/ Acrylated amine synergist (CN3715) (500)	R0101		150	0.32	0.17	5.17
	R0102		150	0.27	0.33	10.98
	R0103		150	0.16	0.11	17.48
	R0104		150	0.54	0.22	4.40
	R0105		150	0.45	0.31	3.09
	R0106		150	0.61	0.69	3.70
3/ Acrylated amine synergist (CN3715) (1000)	R0201		150	0.96	0.59	6.74
	R0202		150	0.45	0.51	12.71
	R0203		150	0.36	0.24	3.01
	R0204		150	0.81	0.40	4.97
	R0205		150	0.23	0.12	5.95
	R0206		150	0.74	0.50	6.67
4/ Acrylated amine synergist (CN3715) (2000)	R0301		150	5.31	3.08	5.21
	R0302		150	0.18	0.12	5.92
	R0303		150	1.01	0.55	5.41
	R0304		150	0.16	0.11	8.54
	R0305		150	0.23	0.19	2.58
	R0306		150	0.36	0.09	14.13
5/ EMS (200)	R0401		150	7.37	0.54	5.08
	R0402		150	20.77	6.24	8.00
	R0403		150	10.59	2.53	6.43
SD		Standard Deviation
EMS		Ethyl Methanesulfonate

 

 

Table 9.9: Duodenum: Animal Comet Data

Group/ Dose Level	Animal		Total	Tail Intensity (%)		Hedgehogs
(mg/kg/day)	Number		Comets	Mean	SD	(%)
1/ Vehicle (0)	R0001		150	0.17	0.09	11.27
	R0002		150	0.14	0.04	15.32
	R0003		150	0.23	0.32	9.74
	R0004		150	0.06	0.03	5.68
	R0005		150	0.13	0.10	5.84
	R0006		150	0.19	0.13	10.26
2/ Acrylated amine synergist (CN3715) (500)	R0101		150	0.10	0.03	11.91
	R0102		150	0.21	0.02	7.85
	R0103		150	0.20	0.03	21.59
	R0104		150	0.19	0.16	10.97
	R0105		150	0.32	0.25	8.33
	R0106		150	0.19	0.14	9.46
3/ Acrylated amine synergist (CN3715) (1000)	R0201		150	0.24	0.17	8.51
	R0202		150	0.45	0.48	5.63
	R0203		150	0.22	0.06	12.87
	R0204		150	0.36	0.20	8.41
	R0205		150	0.45	0.40	11.84
	R0206		150	0.56	0.31	12.42
4/ Acrylated amine synergist (CN3715) (2000)	R0301		150	0.10	0.01	13.60
	R0302		150	0.23	0.19	7.14
	R0303		150	0.20	0.14	10.22
	R0304		150	0.08	0.08	9.06
	R0305		150	0.10	0.07	6.39
	R0306		150	0.18	0.09	11.27
5/ EMS (200)	R0401		150	6.68	1.88	8.94
	R0402		150	5.95	0.58	8.42
	R0403		150	5.41	1.14	22.71
SD		Standard Deviation
EMS		Ethyl Methanesulfonate

 

Applicant's summary and conclusion

Conclusions:

It is concluded that, under the conditions of this comet assay, Acrylated amine synergist (CN3715) did not induce DNA strand breaks in the liver, stomach or duodenum of male Sprague Dawley rats administered up to 2000 mg/kg/day (the maximum recommended dose for in vivo comet studies).

Executive summary:

Acrylated amine synergist (CN3715) was tested for its potential to induce DNA strand breaks in the liver, stomach and duodenum of treated rats. As there was no strand break induction observed in any of the somatic tissues, the gonad was not assessed.

Strain / Species:	Sprague Dawley rats
Vehicle:	Corn oil
Administration route:	Oral by gavage
Dosing regimen:	Two administrations at 0 (Day 1) and 21 hours (Day 2)
Sex:	Male rats only due to the potential analysis of gonadal cells and to ethically minimise animal testing.
Dose levels:	500, 1000 and 2000 mg/kg/day
Maximum dose:	Maximum recommended dose for this type of study
Positive control:	Ethyl methanesulfonate 200 mg/kg, single oral administration at 21 hours (Day 2)
Animals per group:	Six (three for the positive control group)
Dose volume:	10 mL/kg
Clinical signs of toxicity:	There were no clinical observations of toxicity for any animal dosed and no apparent test article related impact on animal bodyweights between Day 1 and Day 2 (percentage change values of +1.0%, +2.1% and +2.0% for 500, 1000 and 2000 mg/kg/day, respectively, compared to +1.1% for the concurrent vehicle control group).
Tissues sampled:	Liver, stomach, duodenum and gonad were sampled on Day 2, equivalent to 24 hours.
Formulation analysis:	Analyses confirmed that 50, 100 and 200 mg/mL formulations were homogenous (0.67-1.17% RSD) and achieved intended concentrations (mean values of 99-100%). No test article was detected in the vehicle control sample.
Clinical Chemistry:	No Acrylated amine synergist (CN3715)-related clinical chemistry changes were recorded.
Histopathology:	No macroscopic or microscopic changes were considered related to Acrylated amine synergist (CN3715).
Exposure:	As dosing was via oral gavage, exposure to the stomach and duodenum was assured. With regards to the liver, there were no clinical observations of toxicity and no clinical chemistry changes or histopathological changes related to the test article to provide indirect evidence of systemic exposure. Publically available data suggest Acrylated amine synergist (CN3715) properties are favourable for absorption following exposure via the oral route. Therefore, while this study did not show effects attributable to indirect systemic exposure to the test article and/or its metabolites, absorption is considered very likely by the sponsor based on the physicochemical properties of Acrylated amine synergist (CN3715).
Assay validity:	The vehicle control data were comparable with the laboratory’s historical vehicle control data ranges. The positive control induced significant increases in %tail intensity in the liver, stomach and duodenum (over the current vehicle control group) that were comparable with the laboratory’s historical positive control data. The data were therefore accepted as valid.

 

There were no dose-related increases in %hedgehogs in liver, stomach or duodenum, thus demonstrating that treatment with Acrylated amine synergist (CN3715) did not cause excessive DNA damage that could have interfered with comet analysis.

Animals treated with Acrylated amine synergist (CN3715) at all doses exhibited group mean tail intensities in the liver that fell within the 95% reference range of the laboratory’s historical vehicle control data. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, and the statistical assessment for dose response was not significant (p > 0.05).

There were two individual animal liver %tail intensity values at 2000 mg/kg/day (R0301, 2.39% and R0303, 3.52%) that exceeded the upper limit of the 95% reference range (1.80%). However, these increases above the concurrent vehicle control group and the 95% reference range were not reproduced in all animals at 2000 mg/kg/day, did not increase the group mean above the 95% reference range and did not contribute to a statistically significant increase for the group compared to the vehicle control group. None of the evaluation criteria for a positive result were met and therefore, these isolated data were considered of no biological relevance.

Animals treated with Acrylated amine synergist (CN3715) at all doses exhibited group mean tail intensities in the stomach that fell within the laboratory's the 95% reference range of the laboratory’s historical vehicle control data. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control. There was a statistically significant dose response noted, however, this response was driven predominantly by an isolated animal response at 2000 mg/kg/day (R0301). All individual animal stomach %tail intensity values fell within the 95% reference range, however the %tail intensity for animal R0301 (5.31%) was elevated compared to all other vehicle and test article dosed animals (0.08-1.01%). As it was within the 95% reference range, it fell within the normal variation of the assay and was likely induced by the mechanical processing of the tissue. Overall, it was considered that this isolated animal value, which increased the group mean and contributed to the statistically significant dose response, but was not reproduced in other animals and did not contribute to a statistically significant increase in tail intensity at 2000 mg/kg/day compared to the vehicle control group, was of no biological relevance.

Animals treated with Acrylated amine synergist (CN3715) at all doses exhibited group mean tail intensities in the duodenum that fell within, or marginally below, the 95% reference range of the laboratory’s historical vehicle control data. There was a statistically significant increase in tail intensity at the intermediate dose (1000 mg/kg/day) group only, compared to the concurrent vehicle control; both the low dose (500 mg/kg/day) and high dose (2000 mg/kg/day) groups were not statistically significantly increased compared to the concurrent vehicle control. As all individual animal and group mean %tail intensity values fell within, or marginally below, the 95% reference range (moreover all values fell below the mean value of the laboratory’s historical vehicle control data indicate value fell towards the lower end of normal variation within the assay), and there was no evidence of a dose response, the statistical significance associated with the intermediate dose group was considered of no biological relevance.

These data were considered negative in the liver, stomach and duodenum. Therefore, no analysis of gonad slides is required.

It is concluded that, under the conditions of this comet assay, Acrylated amine synergist (CN3715) did not induce DNA strand breaks in the liver, stomach or duodenum of male Sprague Dawley rats administered up to 2000 mg/kg/day (the maximum recommended dose for in vivo comet studies).