Sodium 1,4-diisodecyl sulphonatosuccinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2020 to 21 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

1

Reference substance name:

sodium bis[C12-14 (branched) alkyl] sulfosuccinate

IUPAC Name:

sodium bis[C12-14 (branched) alkyl] sulfosuccinate

Test material form:

other: White viscous solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL: see confidential details

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL: see confidential details

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was formulated in the selected vehicle (solvent) to provide a suitably concentrated stock solution as follows. The necessary amount of test item was weighed into a calibrated volumetric flask (no correction for purity of the test item was applied). Approximately 80% of the required volume of vehicle (solvent) was added and the formulation was stirred until homogeneity was reached (It was ultrasonicated 2x10 minutes), then the volume was adjusted to the required final level. From the stock solution, several dilutions were prepared using the selected vehicle (solvent) to prepare dosing solutions for lower doses. The vehicle (solvent) were filtered sterile using a 0.22 µm filter (Supplier: Millipore, Lot No.: MP183904G2, Expiry date: September 2021) before the preparation of the dosing formulations in each case. The stock solutions as well as all dilutions (dosing solutions) were prepared freshly at the beginning of the experiments in the testing laboratory in a sterile hood.
- Final dilution of a dissolved solid, stock liquid or gel: All dilutions (dosing solutions) were prepared freshly at the beginning of the experiments in the testing laboratory in a sterile hood.

OTHER SPECIFICS: see confidential datils

Method

Species / strain

Species / strain / cell type:

lymphocytes: peripheral blood

Remarks:

Blood was collected from healthy adult, non-smoking, volunteers (aged 18 to 35 years).

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium or lithium heparin. Immediately after blood collection lymphocyte cultures were started.
- Suitability of cells:
- Normal cell cycle time (negative control):

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 (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.
- Mitogen used for lymphocytes:

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
*Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heatinactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/ streptomycin (50 U/mL and 50 μg/mL respectively) and heparin (30 U/mL).
*Lymphocytes cultures: Whole blood (0.4 mL) treated with heparin will be added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL phytohaemagglutinin (9 mg/mL) was added.

Cytokinesis block (if used):

Cytochalasin B (5 μg/mL), 24 hours incubation (1.5 times normal cell cycle)

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 was obtained from Trinova Biochem GmbH, Giessen, Germany and was 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 body weight). S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES. The above solution was filter (0.22 m)-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. 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 phytohaemagglutinin (9 mg/mL)). The concentration of the S9-fraction in the exposure medium is 1.8% (v/v).

Test concentrations with justification for top dose:

Assay 1 (3 h exposure with and without S9): 0, 78, 156, 313 µg/mL culture medium. The test item precipitated in the culture medium at the highest dose level.
Assay 2 (24 h exposure without S9): 100, 150, 200 and 215 µg/mL culture medium. The highest dose level was chosen based on cytotoxicity (level of necrosis).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
The vehicle of the test item was dimethyl sulfoxide (DMSO).
Solvent for the positive controls: Hanks’ Balanced Salt Solution (HBSS) without calcium and magnesium.

- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in dimethyl sulfoxide at concentrations of 250 mg/mL and below. The stock solution was treated with ultrasonic waves until the test item had completely dissolved. Test item concentrations were used within 1 hour after preparation.

- Justification for percentage of solvent in the final culture medium: The final concentration of the solvent in the culture medium was 1.0% (v/v).

Controls

Negative solvent / vehicle controls:

yes

Remarks:

The negative control used in the test system was the vehicle for the test item: DMSO.

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 (The second assay was repeated)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Assay 1: 3 h exposure
Assay 2: 24 h exposure
- Harvest time after the end of treatment (sampling/recovery times):
Assay 1: 27 hours harvest time (3 h exposure + 24 h Cytochalasin B incubation)
Assay 2: 24 h harvest time (24 h exposure together with Cytochalasin B incubation)

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.
Cytochalasin B (5 µg/mL) 24h 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. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately after, ethanol/ acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the cell 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/ether 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 minutes with 6.7% (v/v) Giemsa 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 with 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):
At least 502 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.
Assay 1: duplicate cultures: 1000 binucleated cells scored (except 1culture at 313 µg/mL test concentration with only 517 binucleated cells scored due to precipitation of the test item)
Assay 2: duplicate cultures: 1000 binucleated cells scored (selected concentrations of Cytogenetic Assay 2C; except 1 culture with only 971 binucleated cells scored due to heavy necrosis at 200 µg/mL concentration and another culture with only 502 binucleated cells scored due to heavy necrosis at 215 µg/mL concentration)
- 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)
%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}

CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / Total number of cells

t = test item or control treatment culture
c = vehicle control culture
- Any supplementary information relevant to cytotoxicity: A minimum of 500 (with a maximum deviation of 5%) cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI). Three to four analyzable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. At the 3 hours exposure time, the test item was not cytotoxic and difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium. At the 24 hours exposure time, the highest dose level examined for micronuclei were the cultures that showed moderate to heavy necrosis whereas the level of necrosis of the lowest dose level was approximately the same as the level of necrosis of the solvent control. Also, cultures treated with an intermediate dose level were examined.
In the second cytogenetic assay the cytotoxicity was measured by the presence of lytic cells as evidence of necrosis. The level of necrosis as described in this study is as follows:
-: No necrosis obserced
+ Slight necrosis observed
++ Moderate necrosis observed
+++ Heavy necrosis observed.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
At least 502 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.

Evaluation 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).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

Graphpad Prism version 8.4.2 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were 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, onesided, 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 and without

Genotoxicity:

negative

Remarks:

Assay 1 (3 h exposure)

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Assay 1 (3 h exposure)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Assay 2 (24 h exposure)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

highest dose of 215 µg/mL induced heavy necrosis (Assay 2, 24h exposure)

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

First Cytogenetic Assay

Based on the results of the dose-range finding test the following dose levels were selected for the first cytogenetic assay:

Without and with S9-mix: 78, 156 and 313 µg/mL culture medium

(3 hours exposure time, 27 hours harvest time).

Table 1 shows the cytokinesis-block proliferation index of cultures treated with various test item concentrations or with the positive or negative control items.

 

Table 1. Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with AEROSOL TR-70 E 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		3		2		5
78		-1		4		4		8
156		1		1		4		5
313^3)		22		5		2^2)		7
0.25-C		32		31		21		52^****
0.1 Colch		71		11		8		19^**

 

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		3		4		7
78		1		4		2		6
156		1		3		5		8
313^3)		3		3		3		6
15 CP		66		16		28		44^****

*)  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⁾  517 binucleated cells were scored for the presence of micronuclei (see study plan deviation).

³⁾  The test item precipitated in the culture medium.

 

All dose levels were selected for scoring of micronuclei. Both in the absence and presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei.

Second Cytogenetic Assay

To obtain more information about the possible clastogenicity and aneugenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay:

Without S9-mix: 10, 50, 75, 100, 125, 150, 200 and 250 µg/mL culture medium (24 hours exposure time, 24 hours harvest time).

Table 2 shows the cytokinesis-block proliferation index and the level of necrosis (determined after cytogenetic assay 2C) of cultures treated with various test item concentrations or with the positive or negative control items.

No appropriate dose levels could be selected for scoring of micronuclei since at the concentration of 200 µg/mL not enough cytotoxicity was observed (11%), whereas the next higher concentration of 250 µg/mL was too toxic for scoring (cell lysis).

 

Table 2. Cytokinesis-Block Proliferation Index and Level of Necrosis of Human Lymphocyte Cultures Treated with AEROSOL TR-70 E 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		^1) Necrosis
0		1.69	-	1.74		1.71		0		-
10		1.69	-	1.71		1.70		1		-
50		1.65	-	1.68		1.66		7		-
75		1.66	-	1.70		1.68		5		-
100		1.71	-	1.73		1.72		-1		+
125		1.62	-	1.67		1.65		9		+
150		1.64	-	1.69		1.66		7		++
200		1.62	-	1.65		1.64		11		+++
250		^2)		^2)		^2)		^2)		^2)
0.15 MMC-C		1.34	-	1.39		1.37		49		ND
0.23 MMC-C		1.29	-	1.30		1.30		58		ND
0.05 Colch		1.02	-	1.02		1.02		97		ND

 

^1)-		No necrosis observed
+		Slight necrosis observed
++		Moderate necrosis observed
+++		Heavy necrosis observed
^1)		Cell lysis
ND		Not Determined

Note: All calculations were performed without rounding off.

 

Based on these results (CBPI) the experiment was repeated in cytogenetic assay 2A with the following dose levels:

Without S9-mix: 10, 100, 150, 200, 210, 220, 230, 240 and 250 µg/mL culture medium

(24 hours exposure time, 24 hours harvest time).

Table 3 shows the cytokinesis-block proliferation index and the level of necrosis (determined after cytogenetic assay 2C) of cultures treated with various test item concentrations or with the positive or negative control items.

No appropriate dose levels could be selected for scoring of micronuclei since no dose level was available with a cytotoxicity of 55 ± 5%.

 

Table 3. Cytokinesis-Block Proliferation Index and Level of Necrosis of Human Lymphocyte Cultures Treated with AEROSOL TR-70 E Lyophilized in Cytogenetic Assay 2A

Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration µg/mL		CBPI				Mean CBPI		% cytostasis		^1) Necrosis
0		1.59	-	1.67		1.63		0		-
10		1.59	-	1.62		1.61		4		-
100		1.66	-	1.70		1.68		-9		+
150		1.54	-	1.55		1.55		13		+/++
200		1.56	-	^2)		1.56				++/^2)
210		^2)		^2)		^2)		^2)		^2)
220		^2)		^2)		^2)		^2)		^2)
230		^2)		^2)		^2)		^2)		^2)
240		^2)		^2)		^2)		^2)		^2)
250		^2)		^2)		^2)		^2)		^2)
0.15MMC-C		1.19	-	1.24		1.22		65		ND
0.23 MMC-C		1.22	-	1.24		1.23		63		ND
0.05 Colch		1.00	-	1.01		1.01		99		ND

 

^1)-		No necrosis observed
+		Slight necrosis observed
++		Moderate necrosis observed
+++		Heavy necrosis observed
^2)		Cell lysis
ND		Not Determined

Note: All calculations were performed without rounding off.

 

Based on these results (CBPI) the experiment was repeated in cytogenetic assay 2B with the following dose levels:

Without S9-mix: 10, 100, 150, 170, 190, 210, 230 and 250 µg/mL culture medium

(24 hours exposure time, 24 hours harvest time).

Table 4 shows the cytokinesis-block proliferation index and the level of necrosis (determined after cytogenetic assay 2C) of cultures treated with various test item concentrations or with the positive or negative control items.

No appropriate dose levels could be selected for scoring of micronuclei since at the concentration of 190 µg/mL not enough cytotoxicity was observed (8%), whereas the next higher concentration of 210 µg/mL was too toxic for scoring (cell lysis).

 

Table 4. Cytokinesis-Block Proliferation Index and Level of Necrosis of Human Lymphocyte Cultures Treated with AEROSOL TR-70 E Lyophilized in Cytogenetic Assay 2B

Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration µg/mL		CBPI				Mean CBPI		% cytostasis		^1) Necrosis
0		1.93	-	1.94		1.93		0		-
10		1.87	-	1.90		1.89		5		-
100		1.90	-	1.95		1.92		1		+
150		1.88	-	1.89		1.88		5		+/++
170		1.88	-	1.90		1.89		5		+/++
190		1.85	-	1.86		1.86		8		++/+++
210		^2)		^2)		^2)		^2)		^2)
230		^2)		^2)		^2)		^2)		^2)
250		^2)		^2)		^2)		^2)		^2)
0.15 MMC-C		1.47	-	1.49		1.48		49		ND
0.23 MMC-C		1.42	-	1.46		1.44		53		ND
0.05 Colch		1.00	-	1.00		1.00		100		ND

 

^1)-		No necrosis observed
+		Slight necrosis observed
++		Moderate necrosis observed
+++		Heavy necrosis observed
^2)		Cell lysis
ND		Not Determined

Note: All calculations were performed without rounding off.

 

Based on these results (CBPI), the experiment was repeated in cytogenetic assay 2C with the following dose levels:

Without S9-mix: 100, 150, 180, 185, 190, 195, 200, 205, 210 and 215 µg/mL culture medium (24 hours exposure time, 24 hours harvest time).

Table 5 shows the cytokinesis-block proliferation index and the level of necrosis of cultures treated with various test item concentrations or with the positive or negative control items.

 

Table 5.Cytokinesis-Block Proliferation Index and Level of Necrosis of Human Lymphocyte Cultures Treated with AEROSOL TR-70 E Lyophilized in Cytogenetic Assay 2C
Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration µg/mL		CBPI				Mean CBPI		% cytostasis		^1) Necrosis
0		1.81	-	1.86		1.83		0		-
100		1.85	-	1.89		1.87		-4		-
150		1.78	-	1.85		1.82		2		+
180		1.78	-	1.79		1.78		6		+++
185		1.78	-	1.82		1.80		4		+++
190		1.77	-	1.82		1.80		4		+++
195		1.71	-	1.76		1.74		12		+++
200		1.70	-	1.75		1.72		13		+++
205		1.72	-	1.75		1.74		12		+++
210		1.74	-	1.77		1.76		9		+++
215		1.65	-	1.70		1.67		19		+++
0.15 MMC-C		1.50	-	1.50		1.50		40		ND
0.23 MMC-C		1.40	-	1.43		1.41		51		ND
0.05 Colch		1.01	-	1.02		1.01		99		ND

 

^1)-		No necrosis observed
+		Slight necrosis observed
++		Moderate necrosis observed
+++		Heavy necrosis observed
ND		Not Determined

Note: All calculations were performed without rounding off.

 

The results of the CBPI showed that all concentrations tested were not cytotoxic enough and could not be used for scoring of binucleated cells with micronuclei. However, a large number of lytic cells were observed at a concentration of 180 µg/mL and above which indicates necrosis and therefore cytotoxicity. In the second cytogenetic assay and cytogenetic assay 2A and 2B a concentration of 250 µg/mL, 200 µg/mL and 210 µg/mL showed cell lysis (no cells present on the slides) and these concentrations could not be scored for the presence of micronuclei. The next lower concentrations of 200 µg/mL, 150 µg/mL and 190 µg/mL showed a cytotoxicity below 14%. However, these concentrations showed moderate to heavy necrosis and low numbers of cells present on the slides. In this study the CBPI could not be used as a measure of cytotoxicity and therefore the level of necrosis was used to determine the dose levels that were selected for the scoring of micronuclei. The following concentrations were selected:

Without S9-mix: 100, 150, 200 and 215 µg/mL culture medium

(24 hours exposure time, 24 hours harvest time).

The test item induced a dose dependent, statistically significant increase in the number of binucleated cells with micronuclei. In addition, a dose related trend was observed (Table 6).

 

Table 6. Number Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with AEROSOL TR-70 E Lyophilized in Cytogenetic Assay 2C
Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration (µg/mL)		Cytostasis (%)		Necrosis ^3)		Number of binucleated cells with micronuclei^1)
						1000	1000	2000
						A	B	A+B
0		0		-		0	0	0
100		-4		-		1	0	1
150		2		+		2	1	3^*
200		13		+++		0	3^2)	3^*
215		19		+++		3	2^2)	5^**
0.15-C		40		ND		18	16	34^****
0.05 Colch		99		ND		20	11	31^****

*)  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⁾  971 and 502 binucleated cells were scored for the presence of micronuclei, respectively (see study plan deviation).

^3)-		No necrosis observed
+		Slight necrosis observed
++		Moderate necrosis observed
+++		Heavy necrosis observed
NA		Not applicable

 

Discussion

The ability of the test item to induce micronuclei in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analyzed was selected based on the solubility of the test item in the culture medium (3 hours exposure time) or on toxicity by means of the level of necrosis (24 hours exposure time).

The number of binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database.

The positive control chemicals, mitomycin C, colchicine and cyclophosphamide 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. 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, the test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei.

In the second cytogenetic assay at the 24 hours continuous exposure time, the test item induced a dose dependent, statistically significant increase in the number of binucleated cells with micronuclei. In addition, a dose related trend was observed (p = 0.012). However, the statistically significant increase was caused by the low number of cells with micronuclei in the solvent control cultures. The number of binucleated cells with micronuclei was well within the 95% control limits of the distribution of the historical negative control database at all concentrations tested and was therefore considered not biologically relevant. 

Applicant's summary and conclusion

Conclusions:

In conclusion, this test is valid and that AEROSOL TR-70 E Lyophilized is not clastogenic and aneugenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate AEROSOL TR-70 E 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 guideline.

A batch with purity of 99.42% was tested in the study; the vehicle of the test item was dimethyl sulfoxide.

In the first cytogenetic assay, the test item was tested up to 313 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. The test item precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test item was tested up to 215 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. The highest dose level was chosen based on cytotoxicity (level of necrosis).

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, colchicine and cyclophosphamide 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. 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, the test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei.

In the second cytogenetic assay at the 24 hours continuous exposure time, the test item induced a dose dependent, statistically significant increase in the number of binucleated cells with micronuclei. In addition, a dose related trend was observed (p = 0.012). However, the statistically significant increase was caused by the low number of cells with micronuclei in the solvent control cultures. The number of binucleated cells with micronuclei was well within the 95% control limits of the distribution of the historical negative control database at all concentrations tested and was therefore considered not biologically relevant. 

In conclusion, this test is valid and AEROSOL TR-70 E Lyophilized is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.