2-[[(butylamino)carbonyl]oxy]ethyl acrylate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From: 26 Apr 2021 to 03 Aug 2021

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 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Remarks:

According to Chem VwV-GLP Nr. 5.3/OECD Guidance

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2-[[(butylamino)carbonyl]oxy]ethyl acrylate

EC Number:

264-036-0

EC Name:

2-[[(butylamino)carbonyl]oxy]ethyl acrylate

Cas Number:

63225-53-6

Molecular formula:

C10H17NO4

IUPAC Name:

2-[[(butylamino)carbonyl]oxy]ethyl acrylate

Test animals

Strain:

Wistar

Remarks:

Species: Han

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
Research Models and Services Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 6-10 weeks
- Weight at study initiation: 164-201 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Group housed in Makrolon Type IV, with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum; Supplier: Envigo Teklad Diets, Madison, Wisconsin, United States of America
- Water: tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 45-65%
- Air changes (per hr): at least 8
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 26 April 2021To: 03 August 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals and ability to form a suitable dosing formulation
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was freshly formulated in corn oil. The formulations were prepared at room temperature, and applied to the animals within 15 minutes
after preparation

Duration of treatment / exposure:

Not applicable

Frequency of treatment:

Single dose

Post exposure period:

Pre-Experiment on Toxicity: The animals were treated once orally with the test item and examined for acute toxic symptoms at intervals of approx. 0-1 h, 2-4 h, 5-6 h, 24 h, 30 h, and 48 h after administration of the test item.
Main study: The animals of all dose groups, except the positive control group, were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and/or 48 h after
administration of the test item. Sampling of the bone marrow was conducted at low, mid and high doses at 24 h and then at the high dose only at 48 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Pre experiment on toxicity: 2 males/2 females
Main study: 6 males per dose

Control animals:

yes, concurrent vehicle

other:

Positive control(s):

Cyclophosphamide dissolved in sterile water
- Justification for choice of positive control(s): Identified as a suitable positive control in the guidelines
- Route of administration: oral
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:

The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The nucleated cells were separated from the erythrocytes using the method of Romagna et. al.

Details of tissue and slide preparation:

Tissue preparation: The cell suspensions were passed through a column consisting of α-cellulose and cellulose. The columns were then washed with Hank’s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded.

Slide preparation: A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 4000 polychromatic erythrocytes (PCE) per animal were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per total erythrocytes. The analysis was performed with coded slides.

Bioanalysis was performed (report number S21-02411-L1) by Eurofins Agroscience Services EAG Laboratories GmbH.
Six additional male rats were assigned to 2 groups in order to be treated as satellite animals for plasma sampling. These animals were treated once with the test item at the maximum tolerated dose level, as determined within the range finding experiment. All animals were sampled twice by retroorbital puncture (eyes alternating). A maximum blood volume of 1.5 mL was withdrawn during the first sampling time point. The first sampling was performed under light isoflurane anaesthesia. The second, terminal sampling was performed under deep CO2 anaesthesia, and the animals were humanely euthanized after sampling by cervical dislocation whilst they were are in deep anaesthesia.
Blood sampling scheme:
Group 1: 1st sampling before first treatment '2nd sampling and termination 1 hour after application
Group 2: 1st sampling 0.5 hours after application, 2nd sampling and termination 4 hours after application.
The blood of the animals was collected in tubes containing K3-EDTA. The blood samples were centrifuged at 10’000 rpm for about 5 minutes to obtain plasma samples. The obtained plasma was divided in duplicates of about 0.2 mL each.

Evaluation criteria:

The test substance is classified as positive in the assay if:
a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent
negative control,
b) This increase is dose-related at least at one sampling time when evaluated with an
appropriate trend test, and
c) Any of these results are outside the distribution of the historical negative control data
(e.g., Poisson-based 95% control limits)

The study was considered valid if the following criteria were met:
• the concurrent negative control is considered acceptable for addition to the laboratory historical control database (should ideally be within the 95% control limits of the distribution of the historical negative control database)
• at least 5 animals per group can be evaluated.
• the appropriate number of doses and cells will be analysed.
• PCE to erythrocyte ratio is not less than 20% of the negative control.
• The positive control shows a statistically significant increase of micronucleated PCEs compared to the negative control and is compatible to those in the historical positive control database.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test using the validated statistical program RScript Wilcoxon_2.Rnw. The Holm-Bonferroni Adjustment method was used to correct for the Familywise error rate of multiple comparisons.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: Decreased activity. No substantial differences between sexes in toxicity were observed, so that only male animals were used in the main experiment.
- Evidence of cytotoxicity in tissue analysed: None
- Rationale for exposure: It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg bw as the upper limit for nontoxic test items.
- Harvest times: Three adequately spaced dose levels spaced by a factor of 2 were administered and samples of bone marrow were collected at the central sampling interval 24 h after treatment.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 4000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. Results are shown in Tables 1, 2, 3, 4, 5 and 6
- Ratio of PCE/NCE (for Micronucleus assay): The ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes. Results are shown in Tables 1, 2, 3, 4, 5 and 6
- Appropriateness of dose levels and route: As estimated by a pre-experiment at 2000 mg/kg bw (the maximum guideline-recommended dose) was suitable as highest treatment dose. The animals treated with the test item and the vehicle control did not exhibit any clinical symptoms.
- Statistical evaluation: In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance were near to the value of the vehicle control group as shown in Table 1, 2, 3, 4, 5 and 6.

A linear regression (least squares, calculated using the validated statistical program RScript
LM_v02.Rnw) was performed to assess a possible dose dependent increase of mean micronuclei values. The mean number of micronuclei obtained for the groups treated with the test item was compared to the vehicle control group. A trend is judged as significant whenever the p-value (probability value) is below 0.05. A p-value of 0.1964 was obtained, demonstrating that there was no dose dependent increase of mean micronuclei values.

A bioanalysis of the test item in plasma (phase number S21-02411-L1) was performed. The method had been successfully validated by procedural recovery samples for determination of the test substance with an LOQ of 0.10 mg/L and up to 10 mg/L in rat plasma according to the guidance document SANTE/2020/12830 rev. 1 of the European Commission. With regard to selectivity, accuracy and precision, the analytical method was applied successfully for the analytical set when analysing the samples of the study. In all plasma samples of animals treated with the test substance at a concentration of 2000 mg/kg bw, however, the residues of the test substance were below the Limit of Detection (i.e., <0.03 mg/L). Rapid hydrolysis of the test substance was suspected as the most probable underlying reason for the missing proof of exposure.

Any other information on results incl. tables

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE per 4000 erythrocytes scoring 24 hours after treatment

Table 1: Vehicle Control

Test Group	Dose mg/kg b.w.	Animal No.	Micronuclei in Polychromatic Erythrocytes (PCE)			Evaluation 500 PCE in total Erythrocytes
			No. PCE	No. MN/4000 PCE	% MN	Total No Ery	NCE per total Ery	Ratio PCE/Total Ery
Corn Oil	0	1	4000	11	0.28	771	271	0.649
		2	4000	2	0.05	934	434	0.535
		3	4000	8	0.20	707	207	0.707
		4	4000	12	0.30	812	312	0.616
		5	4000	7	0.18	823	323	0.608
		6	4000	4	0.10	845	345	0.592
		Mean		7.3	0.19	815.3	315.3	0.618
		SD		3.9	0.10	75.8	75.8	0.058

 

Table 2: Test Item - Low Dose Group

Test Group	Dose mg/kg b.w.	Animal No.	Micronuclei in Polychromatic Erythrocytes (PCE)			Evaluation 500 PCE in  total Erythrocytes
			No. PCE	No. MN/4000 PCE	% MN	Total No Ery	NCE per total Ery	Ratio PCE/Total Ery
Dose 1	500	7	4000	5	0.13	801	301	0.624
		8	4000	6	0.15	691	191	0.724
		9	4000	9	0.23	686	186	0.729
		10	4000	9	0.23	725	225	0.690
		11	4000	10	0.25	737	237	0.678
		12	4000	6	0.15	718	218	0.696
		Mean		7.5	0.19	726.3	226.3	0.690
		SD		2.1	0.05	41.6	41.6	0.038

 

 

 

Table 3: Test Item - Medium Dose Group

Test Group	Dose mg/kg b.w.	Animal No.	Micronuclei in Polychromatic Erythrocytes (PCE)			Evaluation 500 PCE in  total Erythrocytes
			No. PCE	No. MN/4000 PCE	% MN	Total No Ery	NCE per total Ery	Ratio PCE/Total Ery
Dose 2	1000	13	4000	7	0.18	682	182	0.733
		14	4000	9	0.23	782	282	0.639
		15	4000	5	0.13	730	230	0.685
		16	4000	12	0.30	751	251	0.666
		17	4000	15	0.38	790	290	0.633
		18	4000	9	0.23	719	219	0.695
		Mean		9.5	0.24	742.3	242.3	0.675
		SD		3.6	0.09	40.6	40.6	0.037

 

Table 4: Test Item - High Dose Group

Test Group	Dose mg/kg b.w.	Animal No.	Micronuclei in Polychromatic Erythrocytes (PCE)			Evaluation 500 PCE in  total Erythrocytes
			No. PCE	No. MN/4000 PCE	% MN	Total No Ery	NCE per total Ery	Ratio PCE/Total Ery
Dose 3	2000	19	4000	9	0.23	739	239	0.677
		20	4000	9	0.23	769	269	0.650
		21	4000	6	0.15	1129	629	0.443
		22	4000	4	0.10	784	284	0.638
		23	4000	14	0.35	910	410	0.549
		24	4000	13	0.33	1184	684	0.422
		Mean		9.2	0.23	919.2	419.2	0.563
		SD		3.9	0.10	193.7	193.7	0.110

 

 

 

Table 5: Positive Control

Test Group	Dose mg/kg b.w.	Animal No.	Micronuclei in Polychromatic Erythrocytes (PCE)			Evaluation 500 PCE in  total Erythrocytes
			No. PCE	No. MN/4000 PCE	% MN	Total No Ery	NCE per total Ery	Ratio PCE/Total Ery
Positive	20	25	4000	55	1.38	767	267	0.652
		26	4000	105	2.63	948	448	0.527
		27	4000	76	1.90	1104	604	0.453
		28	4000	63	1.58	1115	615	0.448
		29	4000	63	1.58	1080	580	0.463
		30	4000	42	1.05	1281	781	0.390
		Mean		67.3	1.69	1049.2	549.2	0.489
		SD		21.6	0.54	174.3	174.3	0.091

 

 

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE per 4000 erythrocytes scoring 48 hours after treatment

Table 6: Test Item - High Dose Group

Test Group	Dose mg/kg b.w.	Animal No.	Micronuclei in Polychromatic Erythrocytes (PCE)			Evaluation 500 PCE in  total Erythrocytes
			No. PCE	No. MN/4000 PCE	% MN	Total No Ery	NCE per total Ery	Ratio PCE/Total Ery
Dose 3	2000	31	4000	15	0.38	625	125	0.800
		32	4000	10	0.25	820	320	0.610
		33	4000	9	0.23	766	266	0.653
		34	4000	10	0.25	963	463	0.519
		35	4000	7	0.18	969	469	0.516
		36	4000	8	0.20	970	470	0.515
		Mean		9.8	0.25	852.2	352.2	0.602
		SD		2.8	0.07	141.3	141.3	0.113

 

Animal Weights

Dose Group	Animal No.	Animal weights before treatment						Animal weights before sacrifice
		Initial Weight [g]	Mean [g]	SD [g]	Range [g]			Initial Weight [g]	Mean [g]	SD  [g]	Range [g]
Vehicle Control 24 h	1	179.0	179.0	5.4	169.3	-	185.2	185.3	186.2	6.3	174.8	-	193.7
	2	185.2						193.7
	3	169.3						174.8
	4	182.6						189.8
	5	179.4						186.3
	6	178.7						187.2
500 mg/kg b.w. 24 h Dose 1	7	191.4	180.6	10.1	165.7	-	191.9	199.8	187.1	9.7	171.5	-	199.8
	8	181.0						186.6
	9	165.7						171.5
	10	191.9						194.6
	11	178.8						186.0
	12	174.5						184.0
1000 mg/kg b.w. 24 h Dose 2	13	187.0	187.8	9.3	175.6	-	200.6	194.9	193.0	10.8	177.9	-	205.7
	14	196.7						203.9
	15	200.6						205.7
	16	175.6						177.9
	17	183.4						184.8
	18	183.8						190.8
2000 mg/kg b.w. 24 h Dose 3	19	169.8	186.6	11.6	169.8	-	196.6	160.8	184.6	16.6	160.8	-	201.9
	20	194.6						201.9
	21	192.3						187.0
	22	192.1						185.8
	23	196.6						201.8
	24	173.9						170.0
Positive Control 24 h	25	182.6	178.7	4.6	170.4	-	183.5	188.7	184.1	6.8	171.2	-	188.7
	26	170.4						171.2
	27	178.7						188.2
	28	178.4						185.7
	29	178.8						182.1
	30	183.5						188.5
2000 mg/kg b.w. 48 h Dose 3	31	193.1	184.8	12.3	164.3	-	198.7	198.5	188.0	11.3	174.6	-	199.4
	32	198.7						199.4
	33	181.9						174.9
	34	179.7						186.4
	35	164.3						174.6
	36	190.8						194.4

Animal Weights

Dose Group	Animal No.	Animal weights before treatment						Animal weights before sacrifice
		Initial Weight [g]	Mean [g]	SD [g]	Range [g]			Initial Weight [g]	Mean [g]	SD  [g]	Range [g]
Vehicle Control 24 h	1	179.0	179.0	5.4	169.3	-	185.2	185.3	186.2	6.3	174.8	-	193.7
	2	185.2						193.7
	3	169.3						174.8
	4	182.6						189.8
	5	179.4						186.3
	6	178.7						187.2
500 mg/kg b.w. 24 h Dose 1	7	191.4	180.6	10.1	165.7	-	191.9	199.8	187.1	9.7	171.5	-	199.8
	8	181.0						186.6
	9	165.7						171.5
	10	191.9						194.6
	11	178.8						186.0
	12	174.5						184.0
1000 mg/kg b.w. 24 h Dose 2	13	187.0	187.8	9.3	175.6	-	200.6	194.9	193.0	10.8	177.9	-	205.7
	14	196.7						203.9
	15	200.6						205.7
	16	175.6						177.9
	17	183.4						184.8
	18	183.8						190.8
2000 mg/kg b.w. 24 h Dose 3	19	169.8	186.6	11.6	169.8	-	196.6	160.8	184.6	16.6	160.8	-	201.9
	20	194.6						201.9
	21	192.3						187.0
	22	192.1						185.8
	23	196.6						201.8
	24	173.9						170.0
Positive Control 24 h	25	182.6	178.7	4.6	170.4	-	183.5	188.7	184.1	6.8	171.2	-	188.7
	26	170.4						171.2
	27	178.7						188.2
	28	178.4						185.7
	29	178.8						182.1
	30	183.5						188.5
2000 mg/kg b.w. 48 h Dose 3	31	193.1	184.8	12.3	164.3	-	198.7	198.5	188.0	11.3	174.6	-	199.4
	32	198.7						199.4
	33	181.9						174.9
	34	179.7						186.4
	35	164.3						174.6
	36	190.8						194.4

 

 

 

 Historical Control Data (Oct 2014 - Dec 2020)

 

Vehicle Controls (%)	Male animals
min	0.025
max	0.750
mean	0.255
95% Ctr. Limit	0.001	0.509
SD	0.127
2x SD	0.254
Range of individual animal micronuclei values*	1 - 30
No° indiv. values	240

 

Positive Controls (%)	Male animals
min	0.450
max	4.525
mean	1.718
95% Ctr. Limit	-0.086	3.522
SD	0.902
2x SD	1.804
Range of individual animal micronuclei values*	18 - 181
No° indiv. values	161

 

*per 4000 Polychromatic Erythrocytes

 

 

 Historical Control Data (Oct 2014 - Dec 2020)

 

Vehicle Controls (%)	Male animals
min	0.025
max	0.750
mean	0.255
95% Ctr. Limit	0.001	0.509
SD	0.127
2x SD	0.254
Range of individual animal micronuclei values*	1 - 30
No° indiv. values	240

 

Positive Controls (%)	Male animals
min	0.450
max	4.525
mean	1.718
95% Ctr. Limit	-0.086	3.522
SD	0.902
2x SD	1.804
Range of individual animal micronuclei values*	18 - 181
No° indiv. values	161

 

*per 4000 Polychromatic Erythrocytes