Butanoic acid, 4-amino-4-oxosulfo-, N-coco alkyl derivs., monosodium salts, compds. with triethanolamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

issued by Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Butanoic acid, 4-amino-4-oxosulfo-, N-coco alkyl derivs., monosodium salts, compds. with triethanolamine

EC Number:

308-662-5

EC Name:

Butanoic acid, 4-amino-4-oxosulfo-, N-coco alkyl derivs., monosodium salts, compds. with triethanolamine

Cas Number:

98171-53-0

IUPAC Name:

Butanoic acid, 4-amino-4-oxosulfo-, N-coco alkyl derivs., monosodium salts, compds. with triethanolamine

Test material form:

solid

Details on test material:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0015567151
- Expiration date of the lot/batch: 11 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0015567151
- Expiration date of the lot/batch: 11 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: not determined

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: suspended in culture medium
- Final dilution of a dissolved solid, stock liquid or gel: Stock formulations of the test item and serial dilutions were made in culture medium.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspended in culture medium

Method

Species / strain

Species / strain / cell type:

lymphocytes: human lymphocytes, primary culture

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood samples were drawn from healthy non-smoking donors not receiving medication.
- Suitability of cells: yes
- Sex, age and number of blood donors if applicable: Blood was collected from a female donor (32 years old) for Experiment I and from a male donor (25 years old) for Experiment II.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Method of maintenace: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver microsomal fraction S9 Mix

Test concentrations with justification for top dose:

Experiment I (with/out S9 mix):
8.0, 14.0, 24.4, 42.8, 74.8, 131, 229, 401, 702, 2105 µg/mL (top dose with regard to the purity (95%))

Experiment II (without S9 Mix):
2.8, 5.0, 8.7, 15.2, 26.7, 46.6, 81.6, 143, 250, 500 µg/mL

Experiment II (without S9 Mix):
15.7, 27.4, 48.0, 84.0, 147, 257, 450 µg/mL

Dose selection of the highest dose was performed considering the occurrence of cytotoxicity and test item precipitation in accordance with OECD Guideline 487.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Culture medium (Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™) was used as vehicle.
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours in Experiment I, 20 hours in Experiment II (without S9 Mix), 4 hours in Experiment II (with S9 Mix)

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The harvested cells were spun down by gentle centrifugation, re-suspended in "saline G", spun down once again by centrifugation and resuspended in 5 mL KCl solution and incubated at 37 °C. Ice-cold fixative mixture of methanol and glacial acetic acid was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
- the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
- no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent contrl

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976)
- The micronuclei have to be stained in the same way as the main nucleus
- The area of the micronucleus should not extend the third part of the area of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

Rationale for test conditions:

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487.
With regard to the purity (95%) of the test item, 2105 µg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 8.0 to 2105 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 42.8 µg/mL and above in the absence of S9 mix and at 131 µg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Clear toxic effects were observed after 4 hours treatment with 401 µg/mL and above in the absence and presence of S9 mix. Considering the toxicity and precipitation data of Experiment I, 500 µg/mL (without S9 mix) were chosen as top concentration in Experiment II. The experimental part with S9 mix was repeated with a top dose of 450 µg/mL due to an increase in micronucleated cells above the historical control data range in Experiment I.

Evaluation criteria:

The micronucleus assay will be considered acceptable if it meets the following criteria:

a) The rate of micronuclei in the solvent controls falls within the historical laboratory control data range.
b) The rate of micronuclei in the positive controls is statistically significant increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.

A test item can be classified as clastogenic and aneugenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.

Statistics:

Chi square test (α < 0.05)

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Precipitation occured in cytotoxic doses and lower doses

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

See Table 2.

Any other information on results incl. tables

Table 2: Summary of results

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells
		in µg/mL	CBPI		in %**
Exposure period 4 hrs without S9 mix
I	40 hrs	Solvent control^1	1.87		1.10
		Positive control^2	1.69	20.7	15.10^S
		14.0	1.92	n.c.	1.00
		24.4^#	1.86	0.3	1.45
		42.8^P^#	1.93	n.c.	1.13
Exposure period 20 hrs without S9 mix
II	40 hrs	Solvent control^1	2.10		0.35
		Positive control^3	1.65	40.7	2.40^S
		81.6	1.91	17.1	0.30
		143	1.84	23.7	0.25
		250^P	1.76	30.6	0.15
Exposure period 4 hrs with S9 mix
I	40 hrs	Solvent control^1	1.93		1.00
		Positive control^4	1.43	54.3	4.10^S
		42.8	1.79	14.7	0.75
		74.8^#	1.89	4.1	1.35
		131^P	1.84	9.7	1.00
II	40 hrs	Solvent control^1	2.11		0.40
		Positive control^5	1.64	42.2	4.10^S
		84.0	2.01	9.6	0.80
		147	2.16	n.c.	0.30
		257^P	1.74	33.6	0.40

*      For the positive control groups and the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

^#       The number of micronucleated cells was determined in a sample of 4000 binucleated cells

^P       Precipitation occurred at the end of treatment

^S       The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c.  Not calculated as the CBPI is equal or higher than the solvent control value

¹       Culture Medium
²           MMC              0.8 µg/mL

³           Demecolcine   100 ng/mL

⁴           CPA              17.5 µg/mL

⁵           CPA              12.5 µg/mL

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, the test material is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.

Executive summary:

The test material, suspended in culture medium, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed according to Table 1.

In each experimental group two parallel cultures were analyzed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2105 µg/mL of the test item) was chosen with regard to the purity (95%) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487.

The results are summarized in Table 2.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation.

In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment I in the absence of S9 mix, however, two values (1.45 and 1.13%) after treatment with 24.4 and 42.8 µg/mL, respectively, slightly exceeded the historical control data range (0.08 – 1.12% micronucleated cells). In the presence of S9 mix one value (1.35%), after treatment with 74.8 µg/mL, slightly exceeded the historical control data range (0.16 – 1.08% micronucleated cells). None of these values showed a statistical significance.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, the test material is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.