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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A key Ames bacterial mutagenicity test demonstrated that no increases in mutations were observed in 5 different Salmonella typhimurium strains with and without metabolic activation up to cytotoxic concentration for the registered substance.

In a key in vitro mammalian gene mutation test in CHO K1 cells, the registered substance did not induce mutations up to cytotoxic concentrations in the absence and presence of metabolic activation.

In a key in vitro Micronucleus study in cultured peripheral human lymphocytes, the registered substance induced the formation of micronuclei in human lymphocytes after short term (3-hours) exposure. 

Link to relevant study records

Reference

Reference 1

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

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

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.

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.

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

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

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

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.

 

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 October 2020 to 21 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 29 July 2016

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Official Journal L 142, 31/05/2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (Hprt) enzyme locus in CHO K1 Chinese hamster ovary cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO K1

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: The CHO cell line was originally derived from the ovary of a female Chinese hamster (Puck and Kao, 1967). The CHO K1 is a sub-line of CHO cell line. The CHO K1 cell line was purchased from American Type Culture Collection (ATCC).

For cell lines:
- Absence of Mycoplasma contamination: yes: Checking of mycoplasma infection was carried out for each batch of frozen stock; the cell line was tested negative.
- Periodically ‘cleansed’ of spontaneous mutants: yes: Prior to use in this test, the culture was cleansed of pre-existing mutant cells by culturing in HAT medium on 08 April 2018.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
For each experiment, one or more vials were thawed rapidly, the cells were diluted in F12-10 medium (“culture medium”, the content of the medium is listed in Section 5.5.) and incubated at 37°C (±0.5 °C) in a humidified atmosphere (5±0.3% CO2 in air). When cells were growing well, subcultures were established in an appropriate number of flasks. Trypsin-EDTA (0.25% Trypsin, 1 mM EDTA) solution was used for cell detachment to subculture.
Four types of Ham's F12 medium were prepared as follows
1) Final concentration in F12-1:
Foetal bovine serum (FBS, heat inactivated): 1 % v/v
L-Glutamine: 0.01 mL/mL
Antibiotic-Antimycotic solution *0.01 mL/mL
2)Final concentration in F12-5:
Foetal bovine serum (FBS, heat inactivated): 5 % v/v
L-Glutamine: 0.01 mL/mL
Antibiotic-Antimycotic solution *: 0.01 mL/mL
3)Final concentration in F12-10:
Foetal bovine serum (FBS, heat inactivated): 10 % v/v
L-Glutamine: 0.01 mL/mL
Antibiotic-Antimycotic solution *: 0.01 mL/mL
4)Final concentration in F12-SEL**:
Foetal bovine serum (FBS, heat inactivated): 10 % v/v
L-Glutamine: 0.01 mL/mL
Antibiotic-Antimycotic solution *: 0.01 mL/mL

*: Standard content of the antibiotic-antimycotic solution is 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B.
**: Hypoxanthine-free Ham’s F-12 medium was used for preparation of the selection culture medium

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Male Wistar rats (444-628 g animals were 17-20 weeks old at the initiation) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg bw/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed. Initiation date of the induction of liver enzymes used for preparation S9 used in this study was 02 September 2019 (Test Facility internal code: E13142).
On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels) and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized.
Homogenates were centrifuged for 10 minutes at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly and stored at -80 ± 10°C. The date of preparation of S9 fraction for this study was 05 September 2019 (Expiry date: 05 September 2021).
The protein concentration of the preparation was determined by a chemical analyser at 540 nm in the Clinical Chemistry Laboratory of the test Facility. The protein concentration of the S9 fraction used in the study was determined to be 24.5 g/L. The sterility of the preparation was confirmed.

- method of preparation of S9 mix: The S9-mix was prepared as follows:
a) Concentration of the stock solution:
HEPES* 20 mM
KCl 330 mM
MgCl2 50 mM
NADP** 40 mM
D-Glucose 6 phosphate (Monosodium salt) 50 mM
F12-10 -
S9 fraction -
b) Concentration in the mix:
HEPES* 0.2 mL/mL
KCl 0.1 mL/mL
MgCl2 0.1 mL/mL
NADP** 0.1 mL/mL
D-Glucose 6 phosphate (Monosodium salt) 0.1 mL/mL
F12-10 0.1 mL/mL
S9 fraction 0.3 mL/mL
*HEPES = N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**NADP= β-Nicotinamide-adenine dinucleotide-phosphate

Prior to addition to the culture medium the S9-mix was kept in an ice bath.

- concentration or volume of S9 mix and S9 in the final culture medium :
For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to 9 mL of cell culture medium to give a total of 10 mL (the same ratio was applied in those cases when higher treatment volume was used). The final concentration of the liver homogenate in the test system was 3%.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens (2-Aminoanthracene and Benzo(a)pyrene), that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:
Assay 1:
5-hour treatment in the presence of S9-mix: 200, 150, 125, 100, 50 and 25 µg/mL
5-hour treatment in the absence of S9-mix: 40, 30, 20, 10, 5 and 2.5 µg/mL
Assay 2:
5-hour treatment in the presence of S9-mix: 200, 150, 125, 100, 50 and 25 µg/mL
24-hour treatment in the absence of S9-mix: 50, 40, 30, 20, 10 and 5 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous solvents (distilled water )
(DMSO was used as vehicle for the positive controls)

- Justification for choice of solvent/vehicle: The test item was soluble at 200 mg/mL concentration in distilled water. This vehicle (solvent) is compatible with the survival of the cells and the S9 activity.

- Justification for percentage of solvent in the final culture medium:

Untreated negative controls:

yes

Remarks:

HAM F12-1 (5 h -S9); HAM F12-5 (5 h +S9 and 24 h -S9)

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate cultures (in the main tests)
cytotoxicity (plate for survival): triplicate
mutagenicity: for selection of mutants 5 replicate plates; for viability (CE) 3 replicate plates.
- Number of independent experiments: 2 (Assay 1 and Assay 2)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): at least 2x10E6 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Assay 1: 5 hours exposure (with and without metabolic activation)
Assay 2: 5 hours exposure with metabolic activation and 24 hours exposure without metabolic activation
- Harvest time after the end of treatment (plating for survival):
5 hours exposure experiments: 19 h incubation
24 hours exposure experiment: 0 h incubation

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days (end of expression period = Day 8)
- Selection time (if incubation with a selective agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells):
Survival: 7 days (1 d treatment or 5 h treatment and 19 h incubation; 5 d colony growing)
Viability: 13 days (1 d treatment or 5 h treatment and 19 h incubation; 7 d expression; 5 d colony growing)
Mutagenicity: 15 days ((1 d treatment or 5 h treatment and 19 h incubation; 7 d expression; 7 days colony growing in selective medium
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
6-thioguanine, 10 µg/mL, 7 days cell exposure for colony growing in selective medium
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
viability: adjust cell number to 4x10E5 cells/mL, then diluted to 40 cells/mL
mutant phenotype: adjust cell number to 4x10E5 cells/mL

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative survival (RS)
Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutant frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x10E6 cells: 5 plates at 4x10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and were expressed as 6-TG resistant mutants per 10E6 clonable cells.

Evaluation criteria:

ASSAY ACCEPTANCE CRITERIA
The assay was considered valid if all of the following criteria were met (based on the relevant guidelines):
1. The mutant frequency in the negative (vehicle/solvent) control cultures was in accordance with the general historical control data.
2. The positive control chemicals induced a statistically significant increase in mutant frequency and should be within the historical data for positive controls.
3. The cloning efficiency of the negative controls was in the range of 60-140% on Day 1 and 70-130% on Day 8; under these conditions an adequate number of cells are analysable.
4. At least four test item concentrations in duplicate cultures were presented.

EVALUATION CRITERIA
The test item was considered to be mutagenic in this assay if the following criteria were met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
5. The historical control range is considered when deciding if the result is positive.
Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data of untreated control samples was taken into consideration if necessary).
According to the relevant OECD 476 guideline, the biological relevance of the results was considered first, statistical significance was not the only determination factor for a positive response.

Statistics:

Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.
The mutant frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x10E6 cells: 5 plates at 4x10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and were expressed as 6-TG resistant mutants per 10E6 clonable cells.
The mutation frequencies were statistically analysed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2010 software (R-squared values were calculated for the log concentration versus the mutation frequency).
In the statistical analysis, negative trends were not considered significant.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: In Assay 1 and 2, there were no large changes in pH after treatment in any cases.
- Data on osmolality: In Assay 1 and 2, there were no large changes in osmolality after treatment in any cases.
- Precipitation and time of the determination:
In Assay 1 and 2, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation.
RANGE-FINDING/SCREENING STUDIES (if applicable):
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary experiment. 5-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix was performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest test concentration in the preliminary test was 5000 µg/mL (the recommended maximum concentration for a UVCB).
Insolubility was detected in the preliminary experiment. The concentrations selected for the main experiments were based on results of the performed Preliminary Toxicity Test according to the OECD No. 476 guideline instructions (up to the cytotoxicity limit).
Six concentrations were selected for the main experiments.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays, and the observed values were in the expected range (5-20 x 10E-6) as shown in the OECD No. 476 guideline.
The positive controls (DMBA in the presence of metabolic activation and EMS in the absence of metabolic activation) gave the anticipated, statistically significant increases in mutation frequency over the controls and were in good harmony with the historical data in all assays.
The cloning efficiencies for the negative (vehicle) controls on Days 1 and 8 were within the target range of 60-140% and 70-130% in all assays.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency
In Assay 1, in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 200, 150 and 125 µg/mL concentrations showed a relative survival of 0%, 22% and 48%). An evaluation was made using data of five concentrations.
In Assay 1, in the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 40 and 30 µg/mL concentration showed a relative survival of 0% and 21%, respectively). An evaluation was made using data of five concentrations.
In Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, marked cytotoxicity of the test item was observed (the highest concentration of 200, 150 and 125 µg/mL showed a relative survival of 0%, 25% and 50%, respectively). An evaluation was made using data of five concentrations.
In Assay 2, in the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 50 µg/mL concentration showed a relative survival of 12%). An evaluation was made using data of four concentrations.
- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
For the 5-hour treatments, at least 2x10E6 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and in case of the positive control at least 2x10E7 cells were placed in flasks and incubated for about approximately 24 hours before treatment at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air). On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 5-hour treatment contained 1% (v/v) serum (F12-1, for treatment without metabolic activation) or 5% (v/v) serum (F12-5, for treatment with metabolic activation). A suitable volume (100 µL) of vehicle (solvent), test item solution or positive control solution was added to the 10 mL final volume (higher volume using the same ratio was applied in those cases when higher than 10 mL final volume was used). In case of experiment with metabolic activation, 1.0 mL of S9-mix was added to the cultures (higher volume using the same ratio was applied in those cases when higher than 10 mL final volume was used). After the 5-hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), the cultures were washed thoroughly with F12-10 medium (culture medium). Then, dishes were covered with appropriate amount of fresh F12-10 medium (10-60 mL) and incubated for 19 hours at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air).
After the 19-hour incubation period, cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x10E5 cells/mL. Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.
For the 24-hour treatment, at least 2x10E6 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and in case of the positive control at least 2x107 cells were placed in flasks and incubated for approximately 24 hours before treatment at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air). On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 24-hour treatment contained 5% serum (F12-5). A suitable volume (100 µL) of vehicle (solvent), test item solution or positive control solution was added to the 10 mL final volume (the same ratio was applied in those cases when higher than 10 mL final volume was used). After the 24 hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x10E5 cells/mL. Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.
Duplicate cultures were used for each treatment. Solubility of the test item in the cultures was visually examined at the beginning and end of the treatments. Measurement of pH and osmolality was also performed after the treatment.
o Number of cells plated in selective and non-selective medium
Following adjustment of the cultures to 2x10E5 cells/mL, samples from these cultures were diluted to 40 cells/mL using F12-10 medium. Five mL suspension (200 cells/dish) per each culture were plated into 3 parallel dishes (diameter was approx. 60 mm). The dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 5 days for colony growing.

Cultures were maintained in dishes for 7 days, during which time the HPRT-mutation was expressed. During this expression period, the cultures were sub-cultured and maintained at 2x10E5 cells/dish twice (whenever possible) (on Days 1, 4, 6 and 8) to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell density was determined by haemocytometer and cells were plated for viability and for selection of the mutant phenotype.

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: See under “Any other information on results incl. tables”
- Negative (solvent/vehicle) historical control data: See under “Any other information on results incl. tables”

Table 1. Survival Results of Assay 1

S9 mix	Treatment period (hours)	Study phase	Test item or control concentration	Total number of colonies	Cloning Efficiency (CE)	Relative Survival (%) on plates
+	5	A1	150 µg/mL	135	0.225	22
			125 µg/mL	588	0.490	48
			100 µg/mL	976	0.813	80
			50 µg/mL	1170	0.975	96
			25 µg/mL	1282	1.068	105
			Negative control	1217	1.014	100
			Negative control for DMBA (DMSO)	1267	1.056	104
			Untreated control	1305	1.088	107
			Positive control (DMBA)	36	0.030	3
-	5		30 µg/mL*	132	0.220	21
			20 µg/mL	990	0.825	79
			10 µg/mL	1304	1.087	104
			5 µg/mL	1257	1.048	100
			2.5 µg/mL	1279	1.066	102
			Negative control	1253	1.044	100
			Negative control for EMS (DMSO)	1330	1.108	106
			Untreated control	1369	1.141	109
			Positive control (EMS)	1103	0.919	88

A1 = Assay 1

+ = in the presence of S9-mix                               DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL

- = in the absence of S9-mix                                  EMS = Ethyl methanesulfonate, 0.4 µL/mL

Negative (vehicle) control =1% (v/v) Distilled water

DMSO = Dimethyl sulfoxide

 

*Note: Only one replicate was evaluated.

 

Table 2. Survival Results of Assay 2

S9 mix	Treatment period (hours)	Study phase	Test item or control concentration	Total number of colonies	Cloning Efficiency (CE)	Relative Survival (%) on plates
+	5	A2	150 µg/mL	351	0.293	25
			125 µg/mL	696	0.580	50
			100 µg/mL	1209	1.008	87
			50 µg/mL	1324	1.103	95
			25 µg/mL	1237	1.031	89
			Negative control	1390	1.158	100
			Negative control for DMBA (DMSO)	1318	1.098	95
			Untreated control	1338	1.115	96
			Positive control (DMBA)	16	0.013	1
-	24		50 µg/mL	140	0.117	12
			40 µg/mL	2640	2.200	222
			30 µg/mL	4388	7.313	737
			20 µg/mL	1354	1.128	114
			10 µg/mL	1216	1.013	102
			5 µg/mL	1228	1.023	103
			Negative control	1190	0.992	100
			Negative control for EMS (DMSO)	1350	1.125	113
			Untreated control	1244	1.037	105
			Positive control (EMS)	89	0.074	7

A2 = Assay 2

+ = in the presence of S9-mix      DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL

- = in the absence of S9-mix        EMS = Ethyl methanesulfonate, 0.4 µL/mL

Negative (vehicle) control =1% (v/v) Distilled water

DMSO = Dimethyl sulfoxide

 

Note: In Assay 2in the absence of S9-mixatconcentration levels of 40 and 30 µg/mLthe total number of survival colonies, the cloning efficiency and the relative survival on plates were higher than the negative control. However, these concentrations were excluded from the evaluation.

 

Table 3. Viability Results of Assay 1

S9 mix	Treatment period (hours)	Study phase	Test item or control concentration	Total number of colonies	Cloning Efficiency (CE)
+	5	A1	150 µg/mL*	569	0.948
			125 µg/mL	1069	0.891
			100 µg/mL	900	0.750
			50 µg/mL	1074	0.895
			25 µg/mL	1030	0.858
			Negative control	1016	0.847
			Negative control for DMBA (DMSO)	1102	0.918
			Untreated control	1135	0.946
			Positive control (DMBA)	1064	0.887
-	5		30 µg/mL*	521	0.868
			20 µg/mL	971	0.809
			10 µg/mL	910	0.758
			5 µg/mL	1008	0.840
			2.5 µg/mL	932	0.777
			Negative control	1077	0.898
			Negative control for EMS (DMSO)	908	0.757
			Untreated control	1115	0.929
			Positive control (EMS)	715	0.596

A1 = Assay 1

+ = in the presence of S9-mix         DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL

- = in the absence of S9-mix            EMS = Ethyl methanesulfonate, 0.4 µL/mL

Negative (vehicle) control =1% (v/v) Distilled water

DMSO = Dimethyl sulfoxide

 

*Note: One replicate was evaluated.

 

Table 4. Viability Results of Assay 2

S9 mix	Treatment period (hours)	Study phase	Test item or control concentration	Total number of colonies	Cloning Efficiency (CE)
+	5	A2	150 µg/mL	1190	0.992
			125 µg/mL	1089	0.908
			100 µg/mL	1149	0.958
			50 µg/mL	1013	0.844
			25 µg/mL	1080	0.900
			Negative control	1128	0.940
			Negative control for DMBA (DMSO)	1074	0.895
			Untreated control	1021	0.851
			Positive control (DMBA)	967	0.806
-	24		50 µg/mL	1146	0.955
			40 µg/mL	1054	0.878
			30 µg/mL	981	0.818
			20 µg/mL	908	0.757
			10 µg/mL	1068	0.890
			5 µg/mL	972	0.810
			Negative control	1033	0.861
			Negative control for EMS (DMSO)	1087	0.906
			Untreated control	999	0.833
			Positive control (EMS)	234	0.195

A2 = Assay 2

+ = in the presence of S9-mix                                     DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL

- = in the absence of S9-mix                                        EMS = Ethyl methanesulfonate, 0.4 µL/mL

Negative (vehicle) control =1% (v/v) Distilled water

DMSO = Dimethyl sulfoxide

 

Table 5. Mutagenicity Results of Assay 1

S9 mix	Treatment period (hours)	Study phase	Test item or control concentration	Total number of colonies	Mutant frequency
+	5	A1	150 µg/mL^#	15	8.0
			125 µg/mL	32	9.0*
			100 µg/mL	24	8.0
			50 µg/mL	29	8.1
			25 µg/mL	26	7.6
			Negative control	23	6.9
			Negative control for DMBA (DMSO)	22	6.1
			Untreated control	27	7.1
			Positive control (DMBA)	3379	952.1**
-	5		30 µg/mL*	9	5.2
			20 µg/mL	29	9.1
			10 µg/mL	24	7.9
			5 µg/mL	23	6.9
			2.5 µg/mL	24	7.8
			Negative control	28	7.9
			Negative control for EMS (DMSO)	22	7.3
			Untreated control	28	7.6
			Positive control (EMS)	1038	435.2**

* = Statistically significant increase (at p< 0.05) compared to the relevant vehicle control

** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control

 

A1 = Assay 1

+ = in the presence of S9-mix                                        DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL

- = in the absence of S9-mix                                           EMS = Ethyl methanesulfonate, 0.4 µL/mL

Negative (vehicle) control =1% (v/v) Distilled water

DMSO = Dimethyl sulfoxide

Mutant frequencies refer to 10⁶clonable cells.

^#Note: One replicate was evaluated.

 

Table 6: Mutagenicity Results of Assay 2

S9 mix	Treatment period (hours)	Study phase	Test item or control concentration	Total number of colonies	Mutant frequency
+	5	A2	150 µg/mL	46	11.5**
			125 µg/mL	25	6.9
			100 µg/mL	36	9.3
			50 µg/mL	25	7.4
			25 µg/mL	19	5.3
			Negative control	24	6.5
			Negative control for DMBA (DMSO)	18	5.1
			Untreated control	23	6.7*
			Positive control (DMBA)	1813	577.1**
-	24		50 µg/mL	21	5.5
			40 µg/mL	25	7.1
			30 µg/mL	18	5.5
			20 µg/mL	20	6.6
			10 µg/mL	23	6.4
			5 µg/mL	29	9.0**
			Negative control	21	6.1
			Negative control for EMS (DMSO)	31	8.5
			Untreated control	26	7.8
			Positive control (EMS)	908	1200.9**

* = Statistically significant increase (at p< 0.05) compared to the vehicle control for the positive control (DMBA).

** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control.

 

A2 = Assay 2

+ = in the presence of S9-mix                                        DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL

- = in the absence of S9-mix                                           EMS = Ethyl methanesulfonate, 0.4 µL/mL

Negative (vehicle) control =1% (v/v) Distilled water

DMSO = Dimethyl sulfoxide

Mutant frequencies refer to 10⁶clonable cells.

 

Table 7. Historical Control Data

(updated on 17 October 2017 using data of GLP studies)

	Mutation frequency (Number of 6-TG resistant mutants per 10^6clonable cells)
	Untreated control
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	18.3	20.7	19.0
standard deviation	15.1	16.4	17.2
minimum	5.1	5.5	3.3
maximum	64.1	55.5	58.0
n	27	13	14
	DMSO control
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	21.8	18.9	18.4
standard deviation	15.9	11.6	14.4
minimum	5.4	6.5	6.8
maximum	57.3	47.4	48.5
n	29	13	14
	Distilled water / Water based vehicle control
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	11.5	9.1	15.5
standard deviation	3.8	3.4	5.6
minimum	6.1	5.2	9.2
maximum	15.8	11.6	20.1
n	6	3	3
	Positive controls
	DMBA	EMS	EMS
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	905.2	445.6	1176.6
standard deviation	562.7	118.6	610.9
minimum	141.2	239.6	363.1
maximum	2119.4	636.6	2449.8
n	27	13	14

DMSO = Dimethyl sulfoxide

DMBA = 7,12-Dimethylbenz[a]anthracene

EMS = Ethyl methanesulfonate

S9+ = in the presence of S9-mix

S9- = in the absence of S9-mix

Conclusions:

In conclusion, no mutagenic effect of ASE 36 (lyophilized) was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.

Executive summary:

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at theHprtlocus to evaluate the potential of ASE 36 (lyophilized) to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The design of this study was based on the Commission Regulation (EC) No. 440/2008 and OECD No. 476 guideline, and the study was performed in compliance with Charles River Laboratories Hungary Kft. standard operating procedures and with the OECD Principles of Good Laboratory Practice.

Distilled water was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:

 

Assay 1

5-hour treatment in the presence of S9-mix:

200, 150, 125, 100, 50 and 25 µg/mL

5-hour treatment in the absence of S9-mix:

40, 30, 20, 10, 5 and 2.5 µg/mL

 

Assay 2

5-hour treatment in the presence of S9-mix:

200, 150, 125, 100, 50 and 25 µg/mL

24-hour treatment in the absence of S9-mix:

50, 40, 30, 20, 10 and 5 µg/mL

 

In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 8 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 8 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.

 

In Assays 1 and 2, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.

 

In Assay 1, in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 200, 150 and 125 µg/mL concentrations showed a relative survival of 0%, 22% and 48%). An evaluation was made using data of five concentrations. Statistically significant increase in the mutation frequency (at p<0.05 level) was observed in this experiment at the concentration of 125 µg/mL, although the observed value was within the general historical control range. Furthermore, the observed mutant frequency (9.0 x 10^-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10^-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment). Therefore, it was concluded as biologically not relevant increase. Overall, this experiment was concluded as negative.

In Assay 1, in the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 40 and 30 µg/mL concentration showed a relative survival of 0% and 21%, respectively). An evaluation was made using data of five concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

 

In Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, marked cytotoxicity of the test item was observed (the highest concentration of 200, 150 and 125 µg/mL showed a relative survival of 0%, 25% and 50%, respectively). An evaluation was made using data of five concentrations. Statistically significant increase in the mutation frequency (at p<0.01 level) was observed in this experiment at the concentration of 150 µg/mL,although the observed value was within the general historical control range. Furthermore, the observed mutant frequency (11.5 x 10^-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10^-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment). Therefore, it was concluded as biologically not relevant increase. Overall, this experiment was concluded as negative.

 

In Assay 2, in the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 50 µg/mL concentration showed a relative survival of 12%). An evaluation was made using data of four concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

 

The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. At least three evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the maximum recommended concentrations or cytotoxic range in each test). The overall study was considered to be valid.

 

In conclusion, no mutagenic effect of ASE 36 (lyophilized) was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test was conducted according to GLP and valid test methods, therefore it is considered relevant, adaquate and reliable for classification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, Current Step 4 version dated November 9, 2011.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Preliminary test: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate
Main test: 0.316, 1.0, 3.16, 10.0, 31.6 and 100 µg test item/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aqua ad iniectabilia
- Justification for choice of solvent/vehicle: The test item was completely dissolved in aqua ad iniectabilia.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide in aqua ad iniectabilia

Remarks:

(10 µg/plate):TA 1535, TA 100, without S9-mix

Positive controls:

yes

Positive control substance:

other: 2-Nitro-fluorene in DMSO

Remarks:

(10 µg/plate): TA 98, without S9-mix

Positive controls:

yes

Positive control substance:

other: 9-Amino-acridine in ethanol, abs.

Remarks:

(100 µg/plate): TA 1537, without S9-mix

Positive controls:

yes

Positive control substance:

other: Mitomycin C in DMSO

Remarks:

(10µg/plate): TA 102, without S9-mix

Positive controls:

yes

Positive control substance:

other: Benzo(a)pyrene in DMSO

Remarks:

(10 µg/plate): TA 98, TA 102, TA 1537, with S9-mix

Positive controls:

yes

Positive control substance:

other: 2-amino-anthracene in DMSO

Remarks:

(2µg/plate ): TA 100, TA 1535, with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
1st independent experiment: in agar (plate incorporation)
2nd independent experiment: preincubation

DURATION
1st independent experiment
- Exposure duration: 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours
2nd independent experiment
- Preincubation period: 20 minutes
- Exposure duration: 20 minutes + 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method:other: Cytotoxicity is evidenced by a reduction in the number of revertant colonies, a clearing or diminution of the background lawn, or the degree of survival of treatment cultures. Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a sparse background lawn.

Evaluation criteria:

The statistical evaluation of the results of the AMES test is still under discussion. In our laboratory, a test item is considered to show a positive response if
-the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared with the vehicle control to at least 2-fold of the vehicle control for TA98, TA100 and TA102 and 3-fold of the vehicle control for TA1535 and TA1537 in both independent experiments;
Or
-a concentration-related increase of the revertants is observed. The Spearman’s rank correlation coefficient is observed.
Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.

Statistics:

The statistical evaluation of the results of the AMES test is still under discussion. In our laboratory, a test item is considered to show a positive response if
-the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared with the vehicle control to at least 2-fold of the vehicle control for TA98, TA100 and TA102 and 3-fold of the vehicle control for TA1535 and TA1537 in both independent experiments;
Or
-a concentration-related increase of the revertants is observed. The Spearman’s rank correlation coefficient is observed.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at concentrations of 100 µg test item/plate and higher. Hence, 100 µg test item/plate were chosen as top concentration for the main study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at concentrations of 100 µg test item/plate and higher. Hence, 100 µg test item/plate were chosen as top concentration for the main study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at concentrations of 100 µg test item/plate and higher. Hence, 100 µg test item/plate were chosen as top concentration for the main study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at concentrations of 100 µg test item/plate and higher. Hence, 100 µg test item/plate were chosen as top concentration for the main study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at concentrations of 100 µg test item/plate and higher. Hence, 100 µg test item/plate were chosen as top concentration for the main study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the present test conditions the test item tested up to a cytotoxic concentration of 100 µg test item/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Executive summary:

The test item was examined in the 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.04 was used in order to correct the purity of the test item to 100%. Aqua ad iniectabilia was used as vehicle control.
Preliminary test
The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were tested. Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at concentrations of 100 µg test item/plate and higher. Hence, 100 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
Main study
Six concentrations of 0.316, 1.0, 3.16, 10.0, 31.6 and 100 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
Cytotoxicity
In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 100 µg test item/plate in all test strains.
Mutagenicity
No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to a cytotoxic concentration of 100 µg test item/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).
The results for the vehicle controls were within the range of historical control data of the laboratory. The positive control items showed a significant increase in the number of revertant colonies compared to the vehicle controls of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.
In conclusion, under the present test conditions the test item tested up to a cytotoxic concentration of 100 µg test item/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A key subacute OECD No. 422 study was conducted with the registered substance in Wistar rats (12/sex/group by oral gavage at 0 (propylene glycol), 15, 45 and 90 mg/kg bw/day.  Five female and five male animals served as positive control for the Mammalian Erythrocyte Micronucleus Test (MNT). No test item-related effect, thus no evidence of genotoxic activity was seen in the Mammalian Erythrocyte Micronucleus Test.

Additional information

Additional information from genetic toxicity in vitro:

Bacterial mutation

- In a key Ames test performed with registered substance , the test item containing 95.8% active ingredient was examined in the 5 Salmonella typhimuriumstrains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (Flügge, 2013a). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was completely dissolved inaqua ad iniectabilia. A correction factor of 1.04 was used in order to correct the purity of the test item to 100%.Aqua ad iniectabiliawas used as vehicle control. In a preliminary test, cytotoxicity was noted at concentrations of 100 µg test item/plate and higher, hence 100 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test. In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, no increase in revertant colony numbers as compared with control counts was observed up to a cytotoxic concentration of 100 µg test item/plate, in any of the 5 test strains.

- In conclusion, negative results were obtained for the bacterial mutagenicity study, tested up to cytotoxic concentration both with and wihtout metbabolic activation.

 

Mammalian mutagenicity

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the Hprt locus to evaluate the potential of ASE 36 (lyophilized) to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix) (Orosz, 2022). The design of this study was based on the Commission Regulation (EC) No. 440/2008 and OECD No. 476 guideline, and the study was performed in compliance with Charles River Laboratories Hungary Kft. standard operating procedures and with the OECD Principles of Good Laboratory Practice. Distilled water was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:

 Assay 1

5-hour treatment in the presence of S9-mix: 200, 150, 125, 100, 50 and 25 µg/mL

5-hour treatment in the absence of S9-mix: 40, 30, 20, 10, 5 and 2.5 µg/mL

 Assay 2

5-hour treatment in the presence of S9-mix: 200, 150, 125, 100, 50 and 25 µg/mL

24-hour treatment in the absence of S9-mix: 50, 40, 30, 20, 10 and 5 µg/mL

In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 8 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 8 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined. In Assays 1 and 2, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.

In Assay 1, in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 200, 150 and 125 µg/mL concentrations showed a relative survival of 0%, 22% and 48%). An evaluation was made using data of five concentrations. Statistically significant increase in the mutation frequency (at p<0.05 level) was observed in this experiment at the concentration of 125 µg/mL, although the observed value was within the general historical control range. Furthermore, the observed mutant frequency (9.0 x 10^-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10^-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment). Therefore, it was concluded as biologically not relevant increase. Overall, this experiment was concluded as negative.

In Assay 1, in the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 40 and 30 µg/mL concentration showed a relative survival of 0% and 21%, respectively). An evaluation was made using data of five concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

In Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, marked cytotoxicity of the test item was observed (the highest concentration of 200, 150 and 125 µg/mL showed a relative survival of 0%, 25% and 50%, respectively). An evaluation was made using data of five concentrations. Statistically significant increase in the mutation frequency (at p<0.01 level) was observed in this experiment at the concentration of 150 µg/mL, although the observed value was within the general historical control range. Furthermore, the observed mutant frequency (11.5 x 10^-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10^-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment). Therefore, it was concluded as biologically not relevant increase. Overall, this experiment was concluded as negative.

In Assay 2, in the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 50 µg/mL concentration showed a relative survival of 12%). An evaluation was made using data of four concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. At least three evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the maximum recommended concentrations or cytotoxic range in each test). The overall study was considered to be valid.

In conclusion, no mutagenic effect of ASE 36 (lyophilized) was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.

 

Chromosome aberration

The registered substance was evaluated 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 (Eurlings, 2022).  The study procedures described in this report are in compliance with the most recent OECD guidelines. 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.

 

Additional information from genetic toxicity in vivo:

In vivo micronucleus test as part of the OECD 422 study

A key subacute OECD No. 422 study was conducted with the registered substance in Wistar rats (12/sex/group by oral gavage at 0 (propylene glycol), 15, 45 and 90 mg/kg bw/day.  Five female and five male animals served as positive control for the Mammalian Erythrocyte Micronucleus Test (MNT). They were treated once with 60 mg/kg bw Cyclophosphamide, administered by intraperitoneal injection approximately 24-hour prior to scheduled necropsy.

Three sets of bone marrow smears for MNT were prepared from all the animals, including the vehicle control and the positive control groups. The bone marrow was collected from the right femur of the rats immediately after euthanasia (the left femur of all the dosed animals was used for routine histopathology, the left femur of positive control animals was discarded).

All groups treated with the test item which had a higher average number of micronuclei compared with the corresponding negative control group were tested for statistical significance using the Kruskal Wallis test. None were statistically significant, giving a negative response. Thus, the test item gave a negative response in all treatment groups, there was no evidence of any genotoxicity.

The positive and negative control results were also compared, but although the average number of micronuclei was slightly increased in the males, and almost double in the females, neither gave a statistically significant increase. However, the positive control animals were treated separately from the main experiment and therefore this outcome does not invalidate the study. The positive control values are within the historical control range (males 1 – 43, females 5 – 162). It was also noted that the ratio of PCE:NCE/1000 erythrocytes was also lower than the corresponding negative control group, indicating cytotoxicity in the positive control animals. This is generally associated with cell cycle delay and can result in cells with micronuclei having insufficient time to develop into PCEs in the positive controls.

Summary of the micronucleus test (micronucleated PCE/4000 PCE)

	Males	Females
Negative control (vehicle)	10.3 ± 4.6	11.8 ± 4.3	NS
15 mg/kg bw/day	8.7 ± 4.7	10.1 ± 4.3	NS
45 mg/kg bw/day	10.1 ± 4.1	11.9 ± 6.97	NS
90 mg/kg bw/day	12.9 ± 7.4	12.2 ± 3.6	NS
Positive control	13.8 ± 5.1	22.2 ± 13.1	NS

 

Conclusion

Standard information requirements according to REACH Guidance Part 3 R7a were fulfilled for genotoxicity testing, including bacterial and mammalian mutagenicity and chromosomal aberration. Based on the available results, there were no indications of mutagenicity in bacterial or mammalian cells. A positive result was obtained in the in vitro micronucleus assay, however, this was not confirmed in the in vivo micronucleus assay in rats as part of the OECD 422 study, and no further testing is considered needed.  

Justification for classification or non-classification

Based on these results and according to the EC Directive (No.93/21/EEC) and CLP (No. 1272/2008 of 16 December 2008), the test substance does not have to be classified and has no obligatory labelling requirement for genetic toxicity.