Butanedioic acid, 2-sulfo-, mono(C16-18 and C18-unsatd. alkyl) esters, ammonium sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 October 2020 to 1 February 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 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Butanedioic acid,sulfo-, mono (C16-18 and C18-unsatd. alkyl) ester, ammonium sodium salt

EC Number:

604-617-1

Cas Number:

147993-66-6

IUPAC Name:

Butanedioic acid,sulfo-, mono (C16-18 and C18-unsatd. alkyl) ester, ammonium sodium salt

Test material form:

solid: flakes

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Cultured peripheral human lymphocytes: Whole blood samples obtained from young healthy non-smoking volunteers (aged 18 to 35 years) were treated with an anti-coagulant (heparin) and cultured in the presence of a mitogen (phytohaemagglutinin).
- Suitability of cells: These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic and aneugenic activity of a broad range of chemicals.
- Normal cell cycle time (negative control):
Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2020) are presented below:
Dose-range finding study: age 29, AGT = 14.6 h
First cytogenetic assay: age 32, AGT = 13.7 h
Second cytogenetic assay: age 27, AGT = 14.3 h

For lymphocytes:
- Sex, age and number of blood donors: Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years).
- Whether whole blood or separated lymphocytes were used: Whole blood was used
- Whether blood from different donors were pooled or not: not provided
- Mitogen used for lymphocytes: phytohaemagglutinin (Remel Europe Ltd., Dartford, United Kingdom)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
• Culture medium: Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
• Lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel Europe Ltd., Dartford, United Kingdom) was added.
• Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 41 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.0 - 38.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
- method of preparation of S9 mix : S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life Technologies).
The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components
0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium : Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Not specified

Vehicle / solvent:

Vehicle(s)/solvent(s) used: aqueous solvents (water or saline or culture medium): The vehicle for the test item was RPMI 1640 medium (culture medium, Life Technologies, Bleiswijk, The Netherlands).

- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item formed a white/yellowish homogenous suspension in RPMI 1640 medium (Life Technologies, Bleiswijk, The Netherlands).

- Justification for percentage of solvent in the final culture medium: Not applicable since RPMI 1640 medium was used as vehicle/solvent.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640 medium

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2 (First and Second cytogenetic assay)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium (RPMI 1640 medium)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
First cytogenetic assay (in the absence and presence of S9-fraction): 3 hours exposure
Second cytogenetic assay (in the absence of S9-mix): 24 hours exposure
- Harvest time after the end of treatment (sampling/recovery times): 27 h (after 3h exposure) or 24 h (after 24 h exposure)
First cytogenetic assay (in the absence and presence of S9-fraction): 27 hours harvest time
Second cytogenetic assay (in the absence of S9-mix): 24 hours harvest time

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
Cytochalasine B (Sigma; 5 µg/mL) 24 hours exposure
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of
96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with Charles River Den Bosch study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. Since the lowest concentration of MMC-C and CP resulted in a positive response the highest concentration was not examined for the presence of micronuclei. Due to cytotoxicity the number of examined binucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase was observed, this has no effect on the study integrity.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
The following criteria for scoring of binucleated cells were used:
•Main nuclei that were separate and of approximately equal size.
•Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
•Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
•Trinucleated, quadranucleated, or multinucleated cells.
•Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
•The diameter of micronuclei should be less than one-third of the main nucleus.
•Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
•Micronuclei should have similar staining as the main nucleus.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cytokinesis-block proliferation index (CBPI index)
- Any supplementary information relevant to cytotoxicity:
A minimum of 500 cells (with a maximum deviation of 5%) per culture were counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}

((No. mononucleate cells)+(2×No. binucleate cells)+(3×No. multinucleate cells))
CBPI = -------------------------------------------------------------------------------
(Total number of cells)

t = test item or control treatment culture
c = vehicle control culture
Three analyzable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. . The highest dose level examined for micronuclei were the cultures that produced 55 ± 5% cytotoxicity. The lowest dose level had little or no cytotoxicity (approximately the same as solvent control). Also cultures treated with an intermediate dose level were examined.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
During or after exposure of the stimulated human lymphocytes to the test item, cells were cultured to allow chromosome or spindle damage to lead to the formation of micronuclei in interphase cells. Micronuclei are small particles consisting of acentric chromosome fragments (clastogenic event) or whole chromosomes (aneugenic event leading to chromosome loss), which are unable to migrate to the poles during the anaphase stage of cell division. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm.
Prior to the mitosis (during or after exposure of the test item) the chemical cytochalasin B was added to the cultures. Cytochalasin B arrests the formation of actin filaments. Consequently, the cell is not able to divide, but nuclear division still continues. In this way, cytochalasin B allows discrimination between cells that have undergone nuclear division (binucleated) and cells that have not (mononucleated).
Cells were harvested, stained and interphase cells (bi-nucleated cells) were analyzed microscopically for the presence of micronuclei. Results from cultures treated with the test item were compared with control (vehicle) treated cultures.
Micronuclei were evaluated in the first post-exposure mitosis (i.e. 24 hours after exposure).
A test item that induces a positive response in this assay is presumed to be a potential clastogenic or aneugenic agent.

Evaluation criteria:

ACCEPTABILITY CRITERIA
An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).

Statistics:

Graphpad Prism version 8.4.2 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
The Chi-square test showed that there are statistically significant differences between one or more of the test item groups and the vehicle control group. Therefore a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

First Cytogenetic Assay 3 h exposure: statistically significant increase in the number of binucleated with micronuclei at 450 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

First Cytogenetic Assay 3 h exposure: statistically significant increase in the number of binucleated with micronuclei at 750 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Second Cytogenetic Assay 24 h exposure: The test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH and the osmolarity of the culture medium containing 4000 µg/mL were recorded. In the Dose-Range Finding Test, the pH of 4000 µg/mL was 7.855 (compared to 7.775 in the solvent control).
- Data on osmolality: The pH and the osmolarity of the culture medium containing 4000 µg/mL were recorded. In the Dose-Range Finding Test, the osmolarity of 4000 µg/mL was 296 mOsm/kg (compared to 298 mOsm/kg in the solvent control).

RANGE-FINDING/SCREENING STUDIES (if applicable):
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose-range finding test. The test item was tested in the absence and presence of S9-mix.
Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test item for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma; 5 µg/mL) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time.
The highest tested concentration was the recommended 5000 µg/mL.
After 3 hours exposure to the test item in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasine B and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.
Cytotoxicity of the test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
A concentration of 5000 µg/mL showed no precipitation in the culture medium and was used as the highest concentration of the test item.
In the dose-range finding test blood cultures were treated with 156, 313, 625, 1250, 2500 and 5000 µg test item/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix. Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
Distribution historical positive control data from experiments performed between November 2017 and November 2020.
*Binucleated cells, -S9 mix, 3 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 46.7
SD: 20.3
n: 96
Lower Control Limit (95% Control Limits): 7
Upper Control Limit (95% Control Limits): 87
*Binucleated cells, -S9 mix, 24 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 41.4
SD: 17.4
n: 98
Lower Control Limit (95% Control Limits): 7
Upper Control Limit (95% Control Limits): 75
*Binucleated cells, +S9 mix, 3 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 33.0
SD: 13.9
n: 105
Lower Control Limit (95% Control Limits): 6
Upper Control Limit (95% Control Limits): 60
SD = Standard deviation
n = Number of observations

- Negative (solvent/vehicle) historical control data:
Distribution historical negative control data from experiments performed between November 2017 and November 2020.
*Binucleated cells, -S9 mix, 3 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 5.8
SD: 4.1
n: 94
Lower Control Limit (95% Control Limits): -2
Upper Control Limit (95% Control Limits): 14
*Binucleated cells, -S9 mix, 24 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 5.5
SD: 4.1
n: 91
Lower Control Limit (95% Control Limits): -3
Upper Control Limit (95% Control Limits): 13
*Binucleated cells, +S9 mix, 3 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 6.2
SD: 4.1
n: 97
Lower Control Limit (95% Control Limits): -2
Upper Control Limit (95% Control Limits): 14
SD = Standard deviation
n = Number of observations

Any other information on results incl. tables

Table 1. Cytokinesis-Block Proliferation Index of Human Lymphocytes Cultures Treated with ASE 36 (lyophilized) in the First Cytogenetic Assay

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI	% cytostasis
0	1.74	-	1.75	1.74	0
100	1.74	-	1.76	1.75	-1
300	1.51	-	1.55	1.53	29
450	1.40	-	1.40	1.40	46
600	1.37	-	1.38	1.38	49
750	1.34	-	1.35	1.35	53
900	1.27	-	1.29	1.28	62
1050	1.26	-	1.28	1.27	64
0.25 MMC-C	1.52	-	1.55	1.53	28
0.38 MMC-C	1.41	-	1.42	1.42	44
0.1 Colch	1.33	-	1.34	1.34	55
With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI	% cytostasis
0	1.84	-	1.86	1.85	0
100	1.75	-	1.76	1.76	11
300	1.51	-	1.53	1.52	39
450	1.40	-	1.40	1.40	53
600	1.40	-	1.44	1.42	50
750	1.37	-	1.38	1.38	56
900	1.33	-	1.34	1.34	61
1050	1.34	-	1.36	1.35	59
15 CP	1.37	-	1.37	1.37	56
17.5 CP	1.40	-	1.40	1.40	53

Note: All calculations were performed without rounding off.

 

Table 2. Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with ASE 36 (lyophilized) in the First Cytogenetic Assay

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei^1)
		1000	1000	2000
		A	B	A+B
0	0	2	3	5
100	-1	3	3	6
300	29	4	6	10
750	53	10	6	16^**
0.25-C	28	17	23	40^****
0.1 Colch	55	15	19	34^****

 

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei^1)
		1000	1000	2000
		A	B	A+B
0	0	2	3	5
100	11	3	3	6
300	39	6	4	10
450	53	6	14	20^**
15 CP	56	16	23	39^****

*)  Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001.

¹⁾   1000 binucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

 

Table 3. Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with ASE 36 (lyophilized) in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI	% cytostasis
0	1.57	-	1.58	1.57	0
50	1.43	-	1.49	1.46	19
100	1.40	-	1.40	1.40	30
150	1.23	-	1.28	1.26	55
175	1.23	-	1.23	1.23	60
200	1.13	-	1.14	1.14	76
225	1.11	-	1.11	1.11	81
250	1.08	-	1.09	1.08	85
275	1.06	-	1.07	1.07	89
300	1.03	-	1.04	1.04	93
0.15 MMC-C	1.23	-	1.25	1.24	58
0.23 MMC-C	1.16	-	1.17	1.17	71
0.05 Colch	1.00	-	1.01	1.01	99

 Note: All calculations were performed without rounding off.

 

Table 4. Number Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with ASE 36 (lyophilized) in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei^1)
		1000	1000	2000
		A	B	A+B
0	0	4	2	6
5	19	2	0	2
100	30	4	1	5
150	55	3	1	4
0.15-C	58	17	13	30^****
0.05 Colch	99	1^2)	3^2)	4^****

*)  Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001.

¹⁾   1000 binucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

²⁾  38 and 81 binucleated cells were scored for the presence of micronuclei, respectively.

 

Table 5. Scoring of Cells with One, Two or More Nuclei of Human Lymphocyte Cultures Treated with ASE 36 (lyophilized) in the First Cytogenetic Assay

Withoutmetabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	Culture	Number of cells with ….nuclei			CBPI
		1	2	3 or more
0	A	167	291	42	1.75
	B	176	279	45	1.74
100	A	163	303	34	1.74
	B	149	320	31	1.76
300	A	249	247	4	1.51
	B	231	264	5	1.55
450	A	299	200	1	1.40
	B	303	195	2	1.40
600	A	316	183	1	1.37
	B	310	189	1	1.38
750	A	330	169	1	1.34
	B	325	173	2	1.35
900	A	363	137	0	1.27
	B	355	145	0	1.29
1050	A	371	129	0	1.26
	B	360	140	0	1.28
0.25 MMC-C	A	242	255	3	1.52
	B	232	263	5	1.55
0.38 MMC-C	A	290	209	1	1.42
	B	294	206	0	1.41
0.1 Colch	A	333	162	5	1.34
	B	343	148	9	1.33

 

Table 5 continued. Scoring of Cells with One, Two or More Nuclei of Human Lymphocyte Cultures Treated with ASE 36 (lyophilized) in the First Cytogenetic Assay

 

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	Culture	Number of cells with ….nuclei			CBPI
		1	2	3 or more
0	A	133	304	63	1.86
	B	141	298	61	1.84
100	A	161	298	41	1.76
	B	167	289	44	1.75
300	A	260	257	3	1.51
	B	242	253	5	1.53
450	A	300	200	0	1.40
	B	300	199	1	1.40
600	A	300	198	2	1.40
	B	283	215	2	1.44
750	A	313	187	0	1.37
	B	309	191	0	1.38
900	A	333	167	0	1.33
	B	334	164	2	1.34
1050	A	330	170	0	1.34
	B	319	181	0	1.36
15 CP	A	313	187	0	1.37
	B	318	179	3	1.37
17.5 CP	A	303	194	3	1.40
	B	301	198	1	1.40

 

Table 6. Scoring of Cells with One, Two or More Nuclei of Human Lymphocyte Cultures Treated with ASE 36 (lyophilized) in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/mL	Culture	Number of cells with ….nuclei			CBPI
		1	2	3 or more
0	A	255	202	43	1.58
	B	267	182	51	1.57
50	A	318	148	34	1.43
	B	285	184	31	1.49
100	A	325	157	22	1.40
	B	339	163	23	1.40
150	A	366	129	5	1.28
	B	387	110	3	1.23
175	A	396	113	2	1.23
	B	387	109	4	1.23
200	A	434	65	1	1.13
	B	432	67	1	1.14
225	A	447	53	0	1.11
	B	446	54	0	1.11
250	A	457	43	0	1.09
	B	460	39	1	1.08
275	A	464	36	0	1.07
	B	471	29	0	1.06
300	A	483	17	0	1.03
	B	479	21	0	1.04
0.15 MMC-C	A	389	118	0	1.23
	B	376	123	1	1.25
0.23 MMC-C	A	413	87	0	1.17
	B	432	80	0	1.16
0.05 Colch	A	498	2	0	1.00
	B	494	6	0	1.01

Table 7. Historical Control Data for in vitro Micronucleus Studies of the Solvent Control

	Binucleated
	-S9 Mix		+S9 mix
	3 hour exposure	24 hour exposure	3 hour exposure
Mean number of micronucleated cells (per 2000 cells)	5.8	5.5	6.2
SD	4.1	4.1	4.1
n	94	91	97
Lower Control Limit (95% Control Limits)	-2	-3	-2
Upper Control Limit (95% Control Limits)	14	13	14

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between November 2017 and November 2020.

 

Table 8. Historical Control Data for in vitro Micronucleus Studies of the Positive Control Substances

	Binucleated			Binucleated
	-S9 Mix (MMC-C)		+S9 mix (CP)	-S9 (Colch)
	3 hour exposure	24 hour exposure	3 hour exposure	3 hour exposure	24 hour exposure
Number of micronucleated cells (per 2000 cells)	46.7	41.4	33.0	29.4	44.5
SD	20.3	17.4	13.9	28.1	85.3
n	96	98	105	93	90
Lower Control Limit (95% Control Limits)	7	7	6	-26	-123
Upper Control Limit (95% Control Limits)	87	75	60	84	212

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between November 2017 and November 2020.

 

Applicant's summary and conclusion

Conclusions:

ASE 36 (lyophilized) induces the formation of micronuclei in human lymphocytes after short term exposure under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate ASE 36 (lyophilized) for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guidelines.

Batch P201570001 of the test item was an off-white to beige flakes, lyophilized solid. The vehicle of the test item was culture medium.

In the first cytogenetic assay, the test item was tested up to 750 and 450 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively.

In the second cytogenetic assay, the test item was tested up to 150 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix.

For both cytogenetic assays, appropriate toxicity was reached at this dose level.

The number of binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. The positive control chemical colchicine produced a statistically significant increase in the number of binucleated cells with micronuclei in at least one experiment. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the first cytogenetic assay, in the absence and presence of S9-mix, the test item did induce a statistically significant increase in the number of binucleated cells with micronuclei. The number of binucleated cells with micronuclei was above the accepted range. Additionally, a statistically significant dose related trend was observed.

In the second cytogenetic assay with a 24 hours continuous exposure time, the test item did not induce a dose dependent, statistically significant increase in the number of binucleated cells with micronuclei. 

In conclusion, this test is valid and ASE 36 (lyophilized) induces the formation of micronuclei in human lymphocytes after short term exposure under the experimental conditions described in this report.