Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, acetates

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro (Mutagenic effects - bacterial): similar to OECD 471; Bacterial reverse mutation assay. Negative. Reliability = 2 (read-across substance).

In Vitro (Clastogenic effects - mammalian): OECD 473; Chromosome aberrations. Negative. Reliability = 2 (read-across substance).

In Vitro (Mutagenic effects – mammalian): OECD 476; Mouse lymphoma assay. Negative. Reliability = 2 (read-across).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Comparable to guideline study with acceptable restrictions: the experiment was not repeated in a 2nd independent assay to confirm the lack of mutagenic effect. Cover page of the report is missing.

Justification for type of information:

The primary component of all substances provide complete coverage of 68334-33-8. 68334-33-8 shares high structural similarity with 61789-80-8, 68783-78-8, 107-64-2, and 112-02-7. As 68334-33-8 is a UVCB its components encapsulate the other substances except for the counter ion (Cl-). In solution, the counter ions will dissociate from the parent structures. Therefore, we are comparing substances of equivalent nature. CAS 107-64-2 represents the C18 boundary of the 61789-80-8. Ignoring the salt component CAS 61789-80-8 is equivalent to CAS 68334-33-8. 68783-78-8 is a worst case of both 68334-33-8 and 61789-80-8 since it is unsaturated and the SP2 carbon-carbon bonds are of higher reactivity and a more likely site of metabolic activation. The primary component of CAS 112-02-7 is a substructure of all the other substances. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

experiment not repeated in a 2nd independent assay to confirm the lack of mutagenic effect.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers of Aroclor 1254 induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

Toxicity study: 0, 4, 20, 100, 500, 2500 and 10000 µg/plate
Mutagenicity study Salmonella strains: 0, 4, 20, 100, 500 and 1000 µg/plate
Mutagenicity study WP2uvrA strain: 0, 4, 20, 100, 500 and 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: See table 1

Details on test system and experimental conditions:

Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2/litre) at 37°C. The amount of bacteria in suspension was checked by nephelometry. Stock cultures were stored at -80°C.

Preliminary toxicity tests were performed with all tester strains using a small number of plates, results were used to determine dose levels for the mutagenicity study. A reduced rate of spontaneously occurring colonies as well as visible thinning of the bacterial lawn (assessed microscopically) were used as indicators for toxicity. In combination with the mutagenicity study, toxicity testing was performed as follows: 0.1 ml test solution was mixed with 0.1 ml of a 10exp-6 dilution of the overnight culture of TA100 and plated with histidine and biotin rich top agar in triplicate. The solvent control was compared with the number of colonies per plate.

For the mutagenicity study, top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6%) agar, 0.5% NaCl) with a 5 ml 1.0 mM histidine and 1.0 mM biotin solution. With E.coli histidine was replaced by tryptophan ((5 ml, 0.5 mM). The following was added (in order) to 2 ml of molton top agar at 45°C: 0.1 ml test compound solution, 0.1 ml overnight nutrient broth culture of bacterial tester strain, 0.5 ml S9 mix or buffer. After mixing the liquid was poured onto a petridish with minimal agar (1.5% agar, Vogel-Bonner E medium with 2% glucose). After incubation for 48 to 72 hours at 37°C in the dark, revertant colonies were counted. All concentrations were plated in triplicate.

Evaluation criteria:

Toxicity was assessed by a thinning of the bacterial lawn. Mutagenicity was assessed by comparing the number of revertant colonies in the treated cultures with those of the controls.

Statistics:

No formal statistical analysis was carried out.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 25000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Sterility of the S9 mix and test compound was confirmed, and postive and negative control cultures gave the expected number of colonies.
The test material was very toxic to the bacterial strains at concentrations of 2500 µg/plate and higher. Thinning of the bacterial lawn and/or a reduction in the number of colonies was observed at 1000, 2500 and 10000 µg/plate. For mutagenicity testing, 1000 µg/plate was chosen as the top dose for Salmonella strains, and 2500 µg/ml was chosen as the top dose for WP2uvrA.

The test material did not cause a significant increase in the number of revertant colonies with any of the tester strains both in the presence and absence of S9 mix.

Conclusions:

Based on the results of this study, the test substance did not show any mutagenic activity in the five Salmonella typhimurium strains tested with and without metabolic activation.

Executive summary:

The potential of the test substance Dioctadecyldimethylammonium chloride (90% active in isopropanol/water) to induce reverse mutation in bacteria was assessed using five strains of Salmonella typhimurium and Escherichia coli in a test similar to OECD guideline 471. The study predates the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose-levels of the test substance to be used for the mutagenicity study . The test item was then tested in one independent experiment both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. The experiment was performed according to the direct plate incorporation method. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The preliminary assay with or without metabolic activation showed that the test substance demonstrated a potent toxicity from 10 000 to 2 500 µg/plate in all Salmonella strains tested. Under these conditions, the dose of 1000 µg/plate was retained as the maximum dose tested for the mutagenicity assay in Salmonella strains. In Escherichia Coli WP2uvrA, the top dose was 2500 µg/plate.

 

In the main experiment, the number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. No precipitate was observed in the petri plates when scoring the revertants at all dose-levels. Without metabolic activation, toxicity was observed at the dose level of 1000 µg /plate for TA 1537, TA 1538, TA 98 and TA 100 strains. In Escherichia Coli WP2uvrA, toxicity was noted at 2500 µg /plate. With metabolic activation, cytotoxicity was limited to the strain TA 1537 which exhibited toxicity at the highest dose-level of 1000 µg/plate. No significant increase in the mean number of revertants was noted in the five Salmonella typhimurium strains and Escherichia Coli tested in the presence of the test substance neither with nor without metabolic activation. It was concluded that Dioctadecyldimethylammonium chloride was not mutagen under the conditions of the study

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

8th March to 14th September 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Although this study is guideline and GLP compliant and would normally be assigned a reliability of 1 (reliable without restrictions), this study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions).

Justification for type of information:

The primary component of all substances provide complete coverage of 68334-33-8. 68334-33-8 shares high structural similarity with 61789-80-8, 68783-78-8, 107-64-2, and 112-02-7. As 68334-33-8 is a UVCB its components encapsulate the other substances except for the counter ion (Cl-). In solution, the counter ions will dissociate from the parent structures. Therefore, we are comparing substances of equivalent nature. CAS 107-64-2 represents the C18 boundary of the 61789-80-8. Ignoring the salt component CAS 61789-80-8 is equivalent to CAS 68334-33-8. 68783-78-8 is a worst case of both 68334-33-8 and 61789-80-8 since it is unsaturated and the SP2 carbon-carbon bonds are of higher reactivity and a more likely site of metabolic activation. The primary component of CAS 112-02-7 is a substructure of all the other substances. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line was stored in liquid nitrogen. The thawed stock cultures were propagated at 37°C in 175cm2 plastic flasks. Seeding was done with about 8-10 x 10E5 cells per flask in 30ml of MEM-medium supplemented with 10% fetal calf serum. The cells were subcultured twice weekly.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix obtained from the liver of male Sprague-Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

3 concentrations were used. The highest concentration did not reduce the number of scorable metaphases more than 20% of the negative controls.
Without metabolic activation, the concentration range was 0, 4, 20 and 40 µg/mL.
With metabolic activation, the concentration range was 0, 5, 25 and 50 µg/mL

Vehicle / solvent:

Cell culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation 2000 µg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation 5 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 4 hours
- Expression time (cells in growth medium): 24 hours
- Selection time (if incubation with a selection agent): Not documented
- Fixation time (start of exposure up to fixation or harvest of cells): 12 hours

SELECTION AGENT (mutation assays): Not documented
SPINDLE INHIBITOR (cytogenetic assays): Colcemide
STAIN (for cytogenetic assays): The following was the process used for staining:
-staining for 10 minutes in 2% orcein solution.
-rinsing 3 times in distilled water
-rinsing twice in acetone
-brief rinsing in acetone/xylene
-2 minutes in acetone/xylene
-5 minutes in xylene
-10 minutes in xylene
-embedding in Entellan or Eukitt.

NUMBER OF REPLICATIONS: Duplicate - 2 independent cell cultures were used.
NUMBER OF CELLS EVALUATED: 100 metaphases per experimental group were examined.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Not documented
- Determination of endoreplication: Not documented
- Other: Not documented

OTHER:
- Preparation of microscope slides: after treatment with spindle inhibitor each culture recieved a hypotonic treatment (KCl 0.075 M). Afterwards cells were fixed in a methanol/acetic acid mixture (3/1; v/v) and spread on glass slides before staining
- All metaphase analyses were performed blind
- Structural aberrations recorded: gaps, chromatid and chromosome breaks and exchanges, multiple aberrations and pulverizations

Evaluation criteria:

The test substance was classified as mutagenic if it induced a significantly increased aberration rate compared to the negative controls with one of the concentrations tested. The significance was obvious either by an enhancement of the rate clearly exceeding the control range or it was proven by adequate statistics. The test substance was classified as mutagenic if there was a reproducible concentration related increase in the aberration rate. The test substance was classified as not mutagenic when it tested negative both with and without metabolic activation.

Statistics:

Binomial statistic with Fisher's exact test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

18 and 28 hours after treatment at 40 µg/ml without S9 mix

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the preliminary study, the highest concentration of test substance in culture medium at which no precipitation was observed was found to be 200 µg/ml. The test substance was cytotoxic to the V79 cells from 50 µg/ml up the the limit of solubility in the absence of S9, and 100 µg/ml up to the limit of solubility in the presence of S9.
In the main study, cytoxicity was observed 18 and 28 hours after treatment at 40 µg/ml without S9. There were no increases in the rate of aberrations observed in test groups compared to negative controls, at any concentration both in the presence and absence of S9. Positive control substances demonstrated a significant increase in aberrations compared to negative controls. The results are summarised in Table 1.

Table 1. Summary of results.

Test group	No. cells analysed	Dose µg/ml	S9 mix	Fixation Interval (h)	Percent aberrant cells
					Incl. gaps	Excl. gaps	Exchanges
Solvent control	200	0	-	7	2.5	0.0	0.0
Praepagen WK	200	40	-	7	0.0	0.0	0.0
Solvent control	200	0	+	7	1.5	0.5	0.0
Praepagen WK	200	50	+	7	2.5	1.5	1.0
Negative Control	200	0	-	18	0.0	0.0	0.0
Solvent Control	200	0	-	18	0.5	0.0	0.0
Positive Control EMS	100	2000	-	18	20.5	20.0	46.5
Praepagen WK	200	4	-	18	1.0	0.5	0.5
Praepagen WK	200	20	-	18	1.5	0.5	0.5
Praepagen WK	200	40	-	18	0.5	0.0	0.0
Negative Control	200	0	+	18	1.0	0.0	0.0
Solvent Control	200	0	+	18	1.5	0.0	0.0
Positive Control CPA	200	5	+	18	19.5	16.0	12.5
Praepagen WK	200	5	+	18	1.0	0.0	0.0
Praepagen WK	200	25	+	18	0.5	0.0	0.0
Praepagen WK	200	50	+	18	1.5	0.5	0.0
Solvent control	200	0	-	28	0.0	0.0	0.0
Praepagen WK	200	40	-	28	3.0	0.5	0.0
Solvent control	200	0	+	28	2.5	0.5	0.0
Praepagen WK	200	50	+	28	2.0	0.0	0.0

Conclusions:

Under these experimental conditions, no statistically significant increase in the frequency of cells with chromosome aberrations was observed, both with and without S9 mix, using a dose range from 4 to 50 µg/ml (maximal practicable concentration regarding the cytotoxicity of the substance towards V79 cells). The test substance did not show any clastogenic activity in the in vitro mammalian chromosome aberration test with V79 chinese Hamster cells.

Executive summary:

The test substance Dioctadecyldimethylammonium chloride (90% active in isopropanol/water) was examined for its genotoxic activity in V79 Chinese Hamster cells. The induction of the chromosome aberrations after in vitro treatment was investigated in the presence and in the absence of S9 mix.

 

A preliminary cytotoxicity experiment was performed in order to select the appropriate dose-levels for the main experiment. The test substance produced a significant cytotoxic effect (reduction of plating efficiency) without metabolic activation from 50 µg/ml up to a concentration of 200 µg/ml which was the limit of solubility.

 

In the main experiment, two independent cell cultures with and without metabolic activation (S9 -mix) were used with the dose-levels of 4, 20 and 40 µg/ml in the absence of metabolic activation and 5, 25 and 50 µg/ml in the presence of metabolic activation.

 

The test substance did not induce increase in the number of metaphases with aberration at any preparation time and dose-level. A cytotoxic effect was observed 18 and 28 hours after treatment at 40 µg/ml without metabolic activation. Marked increases in the rate of chromosome aberrations were observed with the positive controls indicating the sensitivity of the assay. In conclusion, dioctadecyldimethylammonium chloride does not induce chromosome mutations (aberrations) in V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system..

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 2010-01-08 to 2010-05-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Although this study is guideline and GLP compliant and would normally be assigned a reliability of 1 (reliable without restrictions), this study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions).

Justification for type of information:

The primary component of all substances provide complete coverage of 68334-33-8. 68334-33-8 shares high structural similarity with 61789-80-8, 68783-78-8, 107-64-2, and 112-02-7. As 68334-33-8 is a UVCB its components encapsulate the other substances except for the counter ion (Cl-). In solution, the counter ions will dissociate from the parent structures. Therefore, we are comparing substances of equivalent nature. CAS 107-64-2 represents the C18 boundary of the 61789-80-8. Ignoring the salt component CAS 61789-80-8 is equivalent to CAS 68334-33-8. 68783-78-8 is a worst case of both 68334-33-8 and 61789-80-8 since it is unsaturated and the SP2 carbon-carbon bonds are of higher reactivity and a more likely site of metabolic activation. The primary component of CAS 112-02-7 is a substructure of all the other substances. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not indicated

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (liver post-mitochondrial fraction and cofactors of rats induced with Aroclor 1254)

Test concentrations with justification for top dose:

Preliminary test:
-1, 10, 50, 100, 250, 500 µg/mL
Mutagenicity experiments without S9 mix:
- 2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the first experiment (3-hour treatment),
- 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL for the second experiment (24 hour treatment)
Mutagenicity experiements with S9 mix:
- 4.69, 9.38, 18.75, 37.5, 75 and 150 µg/mL for the first experiment (3 hours),
- 2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the second experiment (3 hours)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Soluble in ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix, 25 µg/mL (3h treatment) or 5 µg/mL (24h treatment)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix, 3 µg/mL

Details on test system and experimental conditions:

DURATION
- Exposure duration: first experiment 3h with and without S9 mix; second experiment 3 h with S9 mix and 24h without S9 mix (as results of the first experiment were negative)
- Expression time (cells in growth medium): 48 hours (at 37°C in a humidified atmosphere of 5% CO2/95% air)

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: 2 cultures/dose-level and at least duplicate cultures for the controls

NUMBER OF CELLS EVALUATED: 2 000cells/ well (four 96-well plates/culture = eight plates/dose-level) to select the (trifluorothymidine resistant) mutant cells.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG) ; relative suspension growth (RSG) ; cloning efficiency (CE2)

Evaluation criteria:

IWGT recommendations were followed and a positive result is considered when following criteria are fulfilled :
- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control equals or exceeds the global evaluation factor (126 x 10E-6 for the microtiter method)
- a dose-related trend is demonstrated by a statistically significant trend test

Unless considered as clearly positive, the reproducibility of a positive effect should be confirmed.

Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with RTG between 10 and 20%, is not considered as positive result.

A test item is determined to be non-mutagenic when there is no culture showing an Adj. RTG value between 10-20% if:
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence on mutagenicity in a series of data points between 100 to 20% Adj. RTG
- there is no evidence of mutagenicity in a series of data points between 100 to 25% and there is also a negative data point between 10 and 1% Adj. RTG

Statistics:

None

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 mix = 50 µg/mL at the 3-hour treatment; = 10 µg/mL at the 24-hour treatment with S9 mix = 100 µg/mL at the 3-hour treatment

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:

In the solubility assay, the test item was soluble in the vehicle (ethanol) at 1000 mg/mL. Using a treatment volume of 0.5% (v/v), the final dose-level of 5000 µg/mL showed a strong precipitate. Further assays were performed lowering down the concentration of the stock preparation to reach the lowest precipitating dose-level in the culture medium. Using a stock preparation at 100 mg/ml and the maximal practicable treatment volume of 0.5% (v/v), the final dose-level of 500 µg/mL showed a moderate precipitate. At this dose-level, the pH was approximately 7.4 (7.7 for the vehicle control) and the osmolality was equal to 382 mOsm/kg H2O (399 mOsm/kg H2O for the vehicle control).
Therefore, the dose-levels selected for treatment of the preliminary test were 1, 10, 50, 100, 250 and 500 µg/mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY (PRELIMINARY TEST):
Following the 3-hour treatment without S9 mix, a marked to severe toxicity was induced at dose-levels = 10 µg/mL as shown by a 62-100% decrease in adjusted relative total growth (Adj. RTG).
Following the 24-hour treatment without S9 mix, a severe toxicity was induced at dose-levels = 10 µg/mL as shown by a 65-100% decrease in Adj. RTG.
Following the 3-hour treatment with S9 mix, a severe toxicity was induced at dose-levels = 100 µg/mL as shown by a 73-100% decrease in Adj. RTG.

The cloning efficiencies CE₂and the mutation frequencies of the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid.

 Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

 

Experiments without S9 mix

Using a treatment volume of 100 µL/20 mL (0.5% (v/v)), the selected dose-levels were as follows:

.           2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the first experiment (3-hour treatment),

.           1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL for the second experiment (24-hour treatment).

  

Cytotoxicity

Following the 3-hour treatment, a marked to severe toxicity was induced at dose-levels = 37.5 µg/mL, as shown by a 61-100% decrease in Adj. RTG.

Following the 24-hour treatment, a marked to severe toxicity was induced at dose-levels = 6.25 µg/mL, as shown by a 80-100% decrease in Adj. RTG.

Mutagenicity

Following the 3-hour or the 24-hour treatments, no noteworthy increase in the mutation frequency was noted in comparison to the vehicle control.

Experiments with S9 mix

Using a treatment volume of 100 µL/20 mL, the selected dose-levels were as follows:

.           4.69, 9.38, 18.75, 37.5, 75 and 150 µg/mL for the first experiment,

.           2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the second experiment.

 In the first experiment, a slight to strong precipitate was noted in the culture medium at the end of the 3-hour treatment at dose-levels = 4.69 µg/mL.

Cytotoxicity

In the first experiment, a severe toxicity was induced at dose-levels = 75 µg/mL, as shown by a 88-100% decrease in Adj. RTG.

In the second experiment, a moderate to severe toxicity was induced at dose-levels = 37.5 µg/mL, as shown by a 49-100% decrease in Adj. RTG.

Mutagenicity

In either experiment, no noteworthy increase in the mutation frequency was noted in comparison to the vehicle control.

Conclusions:

Under these experimental conditions, no noteworthy increase in the mutation frequency was observed, both with and without S9 mix. The test substance did not show any mutagenic activity in the in vitro mammalian cell gene mutation test with L5178Y TK+/- mouse lymphoma cells.

Executive summary:

The potential for the test substance to induce mutations at the TK locus, was investigated inL5178Y mouse lymphoma cells. The test substance was tested in two independent experiments, both with and without metabolic activation. Approximately 0.5 x 10⁶(3-hour treatment) or 0.15 x 10⁶(24-hour treatment) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main experiments was based on the level of toxicity (decrease in Adj. RTG), according to the criteria specified in the international guidelines.

 

In the experiments without metabolic activation,the selected dose-levels were as follows:

.  2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the first experiment (3-hour treatment),

.  1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL for the second experiment (24-hour treatment).

Cytotoxicity was observed. Following the 3-hour treatment, a marked to severe toxicity was induced at dose-levels = 37.5 µg/mL, as shown by a 61-100% decrease in Adj. RTG. Following the 24-hour treatment, a marked to severe toxicity was induced at dose-levels = 6.25 µg/mL, as shown by a 80-100% decrease in Adj. RTG.

No noteworthy increase in the mutation frequency was noted in comparison to the vehicle control following the 3-hour or the 24-hour treatments.

In the experiments with metabolic activation,the selected dose-levels were as follows:

.  4.69, 9.38, 18.75, 37.5, 75 and 150 µg/mL for the first experiment,

. 2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the second experiment.

 In the first experiment, a slight to strong precipitate was noted in the culture medium at the end of the 3-hour treatment at dose-levels = 4.69 µg/mL.

Cytotoxicity was observed. In the first experiment, a severe toxicity was induced at dose-levels = 75 µg/mL, as shown by a 88-100% decrease in Adj. RTG. In the second experiment, a moderate to severe toxicity was induced at dose-levels = 37.5 µg/mL, as shown by a 49-100% decrease in Adj. RTG.

In either experiment, no noteworthy increase in the mutation frequency was noted in comparison to the vehicle control.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No genetic toxicity studies with the test substance are available. Therefore, a bacterial reverse mutation assay and chromosome aberration study with DODMAC (CAS 107-64-2), and a mouse lymphoma assay with benzyl-di-C16-18-alkylmethylammonium chloride (CAS 61789-73-9) were used as read across to fulfil the data gap for repeated dose toxicity. The primary component of all substances provide complete coverage of 68334-33-8.  68334-33-8 shares high structural similarity with 61789-80-8, 68783-78-8, 107-64-2, and 112-02-7. As 68334-33-8 is a UVCB its components encapsulate the other substances except for the counter ion (Cl-). In solution, the counter ions will dissociate from the parent structures.  Therefore, we are comparing substances of equivalent nature. CAS 107-64-2 represents the C18 boundary of the 61789-80-8. Ignoring the salt component CAS 61789-80-8 is equivalent to CAS 68334-33-8. 68783-78-8 is a worst case of both 68334-33-8 and 61789-80-8 since it is unsaturated and the SP2 carbon-carbon bonds are of higher reactivity and a more likely site of metabolic activation. The primary component of CAS 112-02-7 is a substructure of all the other substances. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

 

The genotoxic properties of DODMAC (90% active in isopropanol/water) regarding point mutation were evaluated in a bacterial reverse mutation assay (Ames test) in a study similar to OECD Guideline 471. The tester strains Salmonella typhimurium TA100, TA1535, TA1537, TA1538 and TA98, and Escherichia coli strain WP2uvrA were exposed to the test substance in the presence and absence of metabolic activation (S9 mix). In a preliminary toxicity study, the test substance was found to be very toxic to the bacteria at concentrations of 2500 µg/plate and above, therefore 1000 µg/plate was chosen as the top dose for S. typhimurium and 2500 µg/plate was chosen as the top dose for E. coli. The test substance did not cause a dose-related significant increase in the number of revertant colonies in any of the strains tested, both with and without metabolic activation. It was concluded that the test material was not mutagenic under the conditions of the study.

 

The potential genotoxic effect of DODMAC (90% active in isopropanol/water) was tested using Chinese Hamster lung fibroblasts (V79), both in the presence and absence of metabolic activation in the form of S-9 mix according to OECD Guideline 473. The limit of solubility in culture medium was 200 µg/mL. Cytoxicity was observed 18 and 28 hours after treatment at 40 µg/mL without S9 but the test substance did not demonstrate an increase in the number of chromosome aberrations with or without metabolic activation compared to negative controls. Based on these results, it was concluded that the substance did not show any clastogenic activity in the in vitro mammalian chromosome aberration test with V79 Chinese Hamster cells.

 

The potential of 'benzyl-di-C16-18-alkylmethylammonium chloride' to induce mutations at the TK locus, was investigated in L5178Y mouse lymphoma cells according to OECD Guideline 476. The test substance was tested in two independent experiments, both with and without metabolic activation. Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main experiments was based on the level of toxicity. Cytotoxicity was observed following the 3-hour and the 24 -hour treatments both with and without metabolic activation but no noteworthy increase in the mutation frequency was noted in comparison to controls. Based on these results, it was concluded that the substance did not show any mutagenic activity in the in vitro mammalian cell gene mutation test with L5178Y TK+/- mouse lymphoma cells.

Justification for classification or non-classification

Based on the available data for the read-across substance, the test substance does not need to be classified for genetic toxicity according to EU Classification and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.