Registration Dossier

Registration Dossier

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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reliable in vitro data ia available on quaternary ammonium compounds, di-C12 -18 -alkyldimethylammonium, nitrites. An OECD 471 study proved to be negative.

In vitro data on the read-across substance, quaternary ammonium compounds, di-C12 -18 -alkyldimethyl, chlorides, (OECD 471, 473 and 476) also proved negative.

However, in vitro studies on sodium nitrite were positive although conflicting results are reported in vivo. This was the reason that the OECD 471 study was conducted on

quaternary ammonium compounds, di-C12 -18 -alkyldimethylammonium, nitrites.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From November 10, 1995 to March 06, 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read across justification is presented from the structurally analogous quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides to the quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites.

In 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of Quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA·solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded then it would seem that to consider read-across data from quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides to the closely structurally analogous quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites to be equally justifiable.

Furthermore, in certain organic solvents it has been reported that the exchange constants between nitrite and chloride in quaternary ammonium salts (QAS) are approximately equal. [Zhurnal Analiticheskoi Khimii, 2010, Vol. 65, No. 6, pp. 579–584. (E.M. Rakhman’ko, M.S. Markovskaya, L.S. Stanishevskii, Yu.S. Zubenko, A.R. Tsyganov)]


Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Didecyldimethylammonium chloride (DDAC)
- Composition of test material, percentage of components: ca. 40% Didecyldimethylammonium chloride (CAS no.: 7173-51-5) in water

- Physical state: Clear viscous liquid
- Analytical purity: 40.37% active ingredient in water
- Stability: Stable
Target gene:
Not applicable
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
TK (Thymidine kinase ) -/+, not able to grow in Trifluorothimidine medium
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix , from Aroclor 1254 induced (500 mg/kg i.p.) rat livers.
Test concentrations with justification for top dose:
Experiments without S9 mix:
0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL (1st experiment),
0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL (2nd experiment)
Experiments with S9 mix:
0.21, 0.62, 1.85, 5.56, 16.7 and 50 µg/mL (1st experiment),
0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL (2nd experiment).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
DURATION
- Exposure duration: Without S9-mix: 1st experiment: 3 h; 2nd experiment: 24 h
With S9-mix: 1st experiment: 3 h; 2nd experiment: 3 h

NUMBER OF REPLICATIONS: Two plates/dose-level for test and four plates for control

NUMBER OF CELLS EVALUATED: 2000 cells/well
Evaluation criteria:
No data
Statistics:
Not reported
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test:
-The test item was freely soluble in the vehicle (DMSO) at 380 mg/mL (expressed as active item). In the culture medium, the dose-level of 3800 µg/mL showed a marked emulsion. At this dose-level, the pH was approximately 7.9 (7.4 for the vehicle control) and no increase in the osmolality due to the test item was noted. Consequently, with a treatment volume of 200 µL/20 mL (1% v/v) culture medium, the dose-levels for the preliminary toxicity test were: 7.6, 76, 380, 760, 1900 and 3800 µg/mL, both with and without S9.

-A slight to marked emulsion was observed at the end of the treatment period at dose-levels ≥ 760 µg/mL.

-Without S9 mix, the test item was markedly to strongly toxic at dose-levels ≥ 7.6 µg/mL (93-100% decrease in the cloning efficiency immediately after treatment (CE0) and in the relative survival (RS)).

-With S9 mix, the test item was markedly to strongly toxic at dose-levels ≥ 76 µg/mL (97-100% decrease in the CE0 and RS).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Based on the preliminary toxicity test for the selection of the doses, the selected dose-levels for treatment were as follow : All the dose-levels were expressed as active item, taking into account the active material content of 40.37%.

Without S9 mix
- First experiment: 0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL
- Second Experiment: 0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL

After 3 h treatment (first experiment), a slight to strong toxicity was showed by 39-69% decrease in the relative  total  growth (RTG)  at  dose- levels  >= 2.78 µg/mL and 96% decrease in the RS at 5.56 µg/mL.
After 24 h treatment (second experiment), the test item was moderately to strongly toxic at dose-levels>=0.56 µl/mL (as shown mainly by 57 -100% decrease in the RS). No noteworthy increase in the mutation frequency was induced both after 3 and 24 h treatments.

With S9 mix
-First experiment: 0.21, 0.62, 1.85, 5.56 16.7 and 50 µg/mL
-Second Experiment: 0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL

The test item was moderately to markedly toxic at dose-levels between 5 and 7.5 µg/mL. At higher dose-levels, the test item was strongly to completely toxic. No noteworthy increase in the mutation frequencies was induced in both experiments.


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

Conclusions:
Interpretation of results (migrated information): negative

The test material (40% didecyldimethylammonium chloride in water) was considered to be non mutagenic in mouse lymphoma assay.
Executive summary:

A study was conducted to evaluate the potential of the test material (40% didecyldimethylammonium chloride (DDAC) in water) to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells. The study was conducted according to OECD guideline 476 and EU method B. 17 and in accordance with GLP

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

After a preliminary toxicity test, test material was tested in two independent experiments, with and without a metabolic activation system, the S9 mix.

The test item was dissolved in DMSO.Since the test material 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.Test material was tested at a concentration range of 0.07- 5.56 (without S9 mix) and 0.21- 50 (with S9 mix) µg DDAC /mL in the main test.A slight to strong toxicity was induced, depending on the dose-levels and the treatment duration. No noteworthy increase in the mutation frequency was induced.

Under the test conditions, test material (40% DDAC in water) was considered to be non mutagenic in mouse lymphoma assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From April 23, 1990 to May 10, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD guideline 471 and EU Method B.13/14 in compliance with GLP
Justification for type of information:
Read across justification is presented from the structurally analogous quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides to the quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites.

In 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of Quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA·solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded then it would seem that to consider read-across data from quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides to the closely structurally analogous quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites to be equally justifiable.

Furthermore, in certain organic solvents it has been reported that the exchange constants between nitrite and chloride in quaternary ammonium salts (QAS) are approximately equal. [Zhurnal Analiticheskoi Khimii, 2010, Vol. 65, No. 6, pp. 579–584. (E.M. Rakhman’ko, M.S. Markovskaya, L.S. Stanishevskii, Yu.S. Zubenko, A.R. Tsyganov)]

Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
Principles of method if other than guideline:
Not applicabke
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): ARQUAD 2C-75 (ARMOFLOTE 18)
- Physical state: Liquid
- Analytical purity: 76.4 %
- Batch No.: 00963
- Stability under test conditions: Stable under storage conditions, stable for at least 4 in water and DMSO
- Storage condition of test material: Stored at room temperature not below 5 °C in the dark
Target gene:
Histidine: hisC3076, hisD3052 pKM101, hisG46, hisG46 pKM101
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 liver mix obtained from Aroclor 1254 induced male Wistar or Sprague-Dawley rats
Test concentrations with justification for top dose:
Preliminary toxicity test:
1.0, 3.3, 10, 33.3, 100, 333, 1000, 3330, 5000 µg/plate

Experiment 1
with S9-mix
0.33, 1.0, 3.3, 10.0, 33.3 µg/plate
without S9-mix
0.1, 0.33, 1.0, 3.3, 10.0 µg/plate

Experiment 2
with S9-mix
0.33, 1.0, 3.3, 10.0, 33.3 µg/plate
without S9-mix
0.1, 0.33, 1.0, 3.3, 10.0 µg/plate
Vehicle / solvent:
Dimethylsulphoxide (DMSO) of spectroscopic quality
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (TA1535), 9-aminoacridine (TA1537), daunomycine (TA98), methylmethanesulfonate (TA100)
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (all 4)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: until 10^9 cells/mL had been obtained
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: Five different doses of the test substance have been tested in triplicate in each strain. An independentrepeat of the experiment was performed.

NUMBER OF CELLS EVALUATED: All colonies were counted.

DETERMINATION OF CYTOTOXICITY
Method: A preliminary toxicity test was performed with TA100 (with and without S9-mix), 9 concentrations were tested in duplicate. The survival of the TA100 culture was determined by comparing the number of colonies on the plate + test substance with those onthe solvent control plate.
Evaluation criteria:
An Ames test was considered acceptable if it met the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should reasonably fall within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which also reasonably fall within the laboratory
historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times
the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary toxicity range-finding
test with strain TA100 or should extend to 5 mg/plate (active ingredient).

A test substance was considered negative (not mutagenic) i n the Ames test if:
a) The total number of revertants in any tester strain at any concentration was not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance was considered positive (mutagenic)in the Ames test if :
a) It induced at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent
control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 was considered to be not significant. If the test substance showed in the first test only a positive response at one or two
concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other extenuating factors might enter into the final evaluation decision.
Statistics:
Not reported
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(in preliminary test)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(in preliminary test)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(in preliminary test)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(in preliminary test)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
No data

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity study with TA100 showed that survival without S9-mix was not or only slightly reduced up to test substance concentration of 10.0 µg/plate and eliminated at and above 33.3 µg/plate. In the presence of S9-mix the survival of strain TA100
was slightly reduced at a test substance concentration of 33.3 µg/plate and eliminated at and above 100 µg/plate. Based on these data, the test substance was tested up to a concentration of 10.0 µg/plate in the absence of S9-mix and up to 33.3 µg/plate in the presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values fell within the testing laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.


ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

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

Under the test conditions, the test material (containing 76.4 % dicocodimethylammonium chloride) was considered to be non mutagenic in the Ames
Salmonella/microsome assay using tester strains TA1535, TA1537, TA98 and TA100.
Executive summary:

A study was performed to evaluate the mutagenic potential of the test material (76.4 % dicocodimethylammonium chloride) in the Ames Salmonella/microsome plate test. The study was conducted according to OECD guideline 471 and EU Method B.13/14 in compliance with GLP

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test material was tested up to 10.0 µg/plate (active ingredient) in the absence of S9-mix and up to 33.3 µg/plate (active ingredient) in the presence of S9 -mix. The test material did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535; TA1537; TA98 and TA100). These results were confirmed in an independently repeated experiment.

Under the test conditions, the test material (containing 76.4 % dicocodimethylammonium chloride) was considered to be non mutagenic in the Ames Salmonella/microsome assay using tester strains TA1535, TA1537, TA98 and TA100.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Remarks:
Type of genotoxicity: chromosome aberration
Adequacy of study:
weight of evidence
Study period:
From November 27, 2001 to March 05, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to OECD guideline 473 and EU method B. 10 in compliance with GLP
Justification for type of information:
Read across justification is presented from the structurally analogous quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides to the quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites.

In 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of Quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA·solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded then it would seem that to consider read-across data from quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides to the closely structurally analogous quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites to be equally justifiable.

Furthermore, in certain organic solvents it has been reported that the exchange constants between nitrite and chloride in quaternary ammonium salts (QAS) are approximately equal. [Zhurnal Analiticheskoi Khimii, 2010, Vol. 65, No. 6, pp. 579–584. (E.M. Rakhman’ko, M.S. Markovskaya, L.S. Stanishevskii, Yu.S. Zubenko, A.R. Tsyganov)]


Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Name of test material (as cited in study report): Arquad 2.10-50 (Didecyldimethylammonium chloride-DDAC )
- Physical state: Clear white to light yellow viscous liquid
- Composition of test material, percentage of components: Act: 51.3 %, ipa: 20.9 %, free amine + amine HCl 0.6
- Analytical purity: 51.3 % active content (Didecyldimethylammonium chloride CAS no.: 7173-51-5.
- Stability: Stable
- Lot/Batch No.: FP95159
Target gene:
Not applicable
Species / strain / cell type:
other: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
None
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Without S9-mix up to 7.5 µL/mL
With S9-mix up to 24 µL/mL in DMSO
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9 mix
Details on test system and experimental conditions:
Organism/cell type: Cultured peripheral human lymphocytes
Metabolic activation system: S9 mix: Aroclor-1254 induced rat liver SS-mix from adult male Wistar rats, from Charles River Wiga, Sulzfeld, Germany.
Evaluation criteria:
Test was considered as acceptable, if it met the following criteria:
1. The Numbers of chromosome aberrations found in the solvent control cultures should reasonable be within the laboratory historical control.
2. The positive control substance should produce a statistically significant increase in the number of cells with chromosome aberrations
3. A homogeneous response between the replicate cultures is observed
Statistics:
Chi square test for the dose related statistical significance
Key result
Species / strain:
hepatocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
- In the first experiment an increase in the number of aberrant cells was found, only at the 24 h fixation time in the presence of S9-mix and at one concentration only (24 µg/mL).
- Since the type of aberrations abserved were only breaks, the increase was not observed at any other concentration and not confirmed in the second experiment, this finding was considered not to be of biological significance.
- Next to this the number of cells with aberrations were just at the historical control data range.
Remarks on result:
other: Migrated from field 'Test system'.
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Table for Cytogenetic In-Vitro-Test: Chromosomal Analysis

positive

Dose in µg/ml

state mean and standard deviations below

control

Experiment 1

Without S9-mix, 24 h fixation

control

3

4.2

5.6

Mitotic index

64

100

82

73

47

Total aberrations with gaps:

61

1

0

4

0

Total aberrations without gaps:

63

1

0

2

0

With S9-mix, 24 h fixation

pos. control

control

10

18

24

Mitotic index

59

100

81

58

41

Total aberrations with gaps:

53

1

6

0

9

Total aberrations without gaps:

52

0

4

0

9

Without S9-mix, 48 h fixation

pos.

control

5.6

Mitotic index

78

100

52

Total aberrations with gaps:

52

1

5

Total aberrations without gaps:

52

0

3

With S9-mix, 48 h fixation

pos.

control

18

Mitotic index

n.a.

100

49

Total aberrations with gaps:

na

1

6

Total aberrations without gaps:

na

1

4

Experiment 2

Without S9-mix, 24 h fixation

pos.

control

3

4.2

7.5

Mitotic index

58

100

83

61

45

Total aberrations with gaps:

33

2

2

2

8

Total aberrations without gaps:

33

1

1

2

4

With S9-mix, 24 h fixation

pos.

control

3

18

24

Mitotic index

51

100

79

61

49

Total aberrations with gaps:

36

3

4

1

5

Total aberrations without gaps:

36

3

2

1

3

Positve controls: the numbers are based on 150 instead 200 cells evaluated.

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

Under the test conditions, the test material ( containing 51.3 % of didecyldimethylammonium chloride-DDAC) was considered to be non clastogenic in human lymphocytes
Executive summary:

A study was conducted to evaluate the genotoxic potential of the test material (51.3 % didecyldimethylammonium chloride- DDAC) in cultured human lymphocytes. The study was conducted according to OECD guideline 473 and EU method B. 10 in compliance with GLP.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

After a preliminary toxicity test, test material was tested in two independent experiments, with and without a metabolic activation system, the S9- mix.

In the absence of S9-mix test material was tested up to 5.6 µg/mL for a 24 and 48 h fixation time in the first experiment. In the second experiment test material was tested up to 7.5 µg/mL for a 24 h fixation time.

In the presence of S9-mix test material was tested up to 24 µg/mL for 24 h fixation time and up to 18 µg/mL for a 48 h fixation time in the first experiment. In the second experiment it was tested up to 24 µg/mL for a 24 h fixation time.

All the concentrations tested except, 24 µg/mL in the presence and absence of S9- mix at 24 and 48 h fixation period did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations.

Under the test conditions, the test material (containing 51.3 % of didecyldimethylammonium chloride-DDAC) was considered to be non clastogenic in human lymphocytes

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 26 October 2017 to February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine (in S. Tryhimurium); Tryptophan (in E-coli)
Vehicle / solvent:
Anhydrous analytical grade dimethyl sulphoxide (DMSO).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
Test System
The test system was suitably labelled to clearly identify the study number, bacterial strain, test article concentration (where appropriate), positive and vehicle controls, absence or presence of S-9 mix

Mutation Experiments
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites was tested for mutation (and toxicity) in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli (WP2 uvrA) in two separate experiments using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45±1°C:
• 0.1 mL bacterial culture
• 0.1 mL test article solution or control
• 0.5 mL 10% S-9 mix or buffer solution

followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C and protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.
It should be noted that Mutation Experiment 2 data for strain TA98 in the absence of S-9 was obtained from a repeat experiment. In error, 2 concentrations (0.16 µg/plate and 1.6 µg/plate) were not treated in the initial treatments. Therefore, data from less than 5 analysable concentrations was obtained to make an assessment of mutagenicity. Data from the initial treatments are for reference only and included only for qualifying data for strain TA98 in the presence of S-9. This is because the data from the vehicle controls in the absence of S-9 are used to confirm the correct strain characteristics on that day of treatment for that experiment. Also, Mutation Experiment 2 data for strains TA1535 and TA1537 in the presence of S-9 were obtained from repeat experiments. The positive control treatments failed to generate an acceptable response in the initial and subsequent experiment for strain TA1537 treatments. The repeat treatments were performed using the same methodology as those used for the initial treatments and the data are presented as the Mutation Experiment 2 data for these strains.

Treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.
Volume additions for the Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (DMSO) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.

Toxicity Assessment
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response. Where mutation data from fewer than five treatment concentrations was obtained, an evaluation of the mutation data for the study as a whole was made.

Colony Enumeration
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or split in agar affected the accuracy of the automated counter.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:

1. A concentration related increase in revertant numbers was ≥2-fold (in strains TA98, TA100 and WP2 uvrA) or ≥3 fold (in strains TA1535 and TA1537) the concurrent vehicle control values
2. The positive trends/effects described above were reproducible.

The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if either of the above criteria were met.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

: Raw Plate Counts and Calculated Mutagenicity Data, Experiment 1, without S‑9
Strain Compound Conc. Level Mean Standard Deviation Fold Increase Revertant Numbers Per Plate
(µg/plate)
TA98 DMSO - 23.7 5.5 - 21, 20, 30
Quaternary ammonium compounds, di-C12-18- 5 21.3 5.5 0.9 25, 24, 15
alkyldimethyl, nitrites 16 23.7 7.5 1 15, 28, 28
50 - - - - T, - T, - T
160 - - - - T, - T, - T
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
2NF 5 656.7 44.9 27.7 708, 637, 625
             
TA100 DMSO - 116 11.1 - 126, 104, 118
Quaternary ammonium compounds, di-C12-18- 5 104.7 6.1 0.9 106, 110, 98
alkyldimethyl, nitrites 16 82.3 4.9 0.7 88 S, 80 S, 79 S
50 - - - - T, - T, - T
160 - - - - T, - T, - T
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
NaN3 2 475 35.9 4.1 460, 516, 449
             
TA1535 DMSO - 12.7 1.2 - 12, 14, 12
5 12.7 6.7 1 7, 11, 20
Quaternary ammonium compounds, di-C12-18- 16 12.3 2.5 1 10, 12, 15
alkyldimethyl, nitrites 50 - - - - T, - T, - T
160 - - - - T, - T, - T
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
NaN3 2 430.3 23.9 34 423, 457, 411
             
TA1537 DMSO - 13 6.6 - 20, 7, 12
Quaternary ammonium compounds, di-C12-18- 5 12.3 3.5 0.9 12, 16, 9
alkyldimethyl, nitrites 16 10.7 5 0.8 10, 16, 6
50 6.7 2.1 0.5 6, 5, 9
160 - - - - T, - T, - T
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
AAC 50 222.7 32.1 17.1 246, 186, 236
             
WP2uvrA DMSO - 23 3.5 - 21, 21, 27
Quaternary ammonium compounds, di-C12-18- 5 29.7 1.5 1.3 28, 30, 31
alkyldimethyl, nitrites 16 27.3 10 1.2 16, 35, 31
50 24.3 2.1 1.1 26, 22, 25
160 - - - - T, - T, - T
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
NQO 2 425.3 34.1 18.5 443, 386, 447
             

Raw Plate Counts and Calculated Mutagenicity Data, Experiment 1, with S‑9
Strain Compound Conc. Level Mean Standard Deviation Fold Increase Revertant Numbers Per Plate
(µg/plate)
TA98 DMSO - 36 10.8 - 33, 48, 27
Quaternary ammonium compounds, di-C12-18- 5 38.3 2.3 1.1 41, 37, 37
alkyldimethyl, nitrites 16 31.3 2.9 0.9 33, 28, 33
50 35.3 7.6 1 27, 42, 37
160 29 3.6 0.8 33 S, 28 S, 26 S
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
B[a]P 10 324.7 39.8 9 334, 281, 359
             
TA100 DMSO - 106.7 9 - 116, 98, 106
Quaternary ammonium compounds, di-C12-18- 5 106.3 2.5 1 104, 109, 106
alkyldimethyl, nitrites 16 118 7.2 1.1 124, 120, 110
50 100.3 6.4 0.9 93, 105, 103
160 70 7.5 0.7 63 S, 69 S, 78 S
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
AAN 5 1209.3 135.1 11.3 1348, 1078, 1202
             
TA1535 DMSO - 24 6.1 - 28, 27, 17
Quaternary ammonium compounds, di-C12-18- 5 18 6.6 0.8 11, 19, 24
alkyldimethyl, nitrites 16 20.3 1.5 0.8 20, 22, 19
50 17.7 5.9 0.7 22, 20, 11
160 12 2.6 0.5 10 S, 15 S, 11 S
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
AAN 5 243.7 33.2 10.2 282, 225, 224
             
TA1537 DMSO - 9 2 - 11, 9, 7
Quaternary ammonium compounds, di-C12-18- 5 9.3 0.6 1 10, 9, 9
alkyldimethyl, nitrites 16 8.7 4.7 1 7, 14, 5
50 10.7 3.5 1.2 14, 7, 11
160 10.3 3.5 1.1 7 V, 10 V, 14 V
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
AAN 5 366 25 40.7 374, 338, 386
             
WP2uvrA DMSO - 28.3 1.5 - 27, 30, 28
Quaternary ammonium compounds, di-C12-18- 5 32.3 11.6 1.1 19, 40, 38
alkyldimethyl, nitrites 16 33 10.1 1.2 42, 22, 35
50 28.7 6.4 1 24, 36, 26
160 31.7 7.2 1.1 27, 40, 28
500 3.7 1.5 0.1 4 V, 2 V, 5 V
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
AAN 15 203.3 15.1 7.2 186, 214, 210
             

Raw Plate Counts and Calculated Mutagenicity Data, Experiment 2, without S‑9
Strain Compound Conc. Level Mean Standard Deviation Fold Increase Revertant Numbers Per Plate
(µg/plate)
TA98 DMSO - 15.7 5 - 11, 15, 21
Quaternary ammonium compounds, di-C12-18- 0.05 22.7 5.5 1.4 19, 20, 29
alkyldimethyl, nitrites 0.16 - - - - U, - U, - U
0.5 18.3 1.2 1.2 19, 19, 17
1.6 - - - - U, - U, - U
5 25.3 5.7 1.6 27, 19, 30
16 23 2.6 1.5 24, 25, 20
50 - - - - T, - T, - T
2NF 5 1517.7 202.5 96.9 1520, 1314, 1719
             
TA100 DMSO - 92 18.1 - 94, 109, 73
Quaternary ammonium compounds, di-C12-18- 0.05 89.3 7.2 1 81, 94, 93
alkyldimethyl, nitrites 0.16 101.7 15 1.1 87, 117, 101
0.5 89 13.1 1 77, 87, 103
1.6 91 8.9 1 88, 84, 101
5 83.3 5.1 0.9 89, 82, 79
16 67 10.1 0.7 58, 78, 65
50 - - - - T, - T, - T
NaN3 2 887 13 9.6 880, 879, 902
             
TA1535 DMSO - 9.3 3.1 - 12, 10, 6
Quaternary ammonium compounds, di-C12-18- 0.05 6.7 2.9 0.7 5, 5, 10
alkyldimethyl, nitrites 0.16 12.3 4.2 1.3 9, 17, 11
0.5 9 6.6 1 15, 10, 2
1.6 7.3 1.5 0.8 7, 6, 9
5 9.7 2.5 1 7, 12, 10
16 10.3 4.5 1.1 10, 15, 6
50 - - - - T, - T, - T
NaN3 2 702 37.3 75.2 683, 745, 678
             
TA1537 DMSO - 15 6 - 9, 21, 15
Quaternary ammonium compounds, di-C12-18- 0.16 15.7 2.9 1 14, 19, 14
alkyldimethyl, nitrites 0.5 15.3 1.5 1 15, 14, 17
1.6 11.7 0.6 0.8 11, 12, 12
5 11.7 2.5 0.8 12, 14, 9
16 6.7 2.1 0.4 9, 5, 6
50 - - - - T, - T, - T
160 - - - - T, - T, - T
AAC 50 289.3 72.2 19.3 332, 330, 206
             
WP2uvrA DMSO - 21.7 5 - 21, 17, 27
Quaternary ammonium compounds, di-C12-18- 0.16 22.7 6 1 29, 22, 17
alkyldimethyl, nitrites 0.5 22 2.6 1 25, 21, 20
1.6 19 3.5 0.9 15, 21, 21
5 23.3 6 1.1 29, 17, 24
16 17.3 1.5 0.8 16, 17, 19
50 20 4.6 0.9 24, 15, 21
160 - - - - T, - T, - T
NQO 2 513.3 26 23.7 487, 539, 514
             

Raw Plate Counts and Calculated Mutagenicity Data, Experiment 2, with S‑9
Strain Compound Conc. Level Mean Standard Deviation Fold Increase Revertant Numbers Per Plate
(µg/plate)
TA98 DMSO - 43.7 4.9 - 38, 47, 46
Quaternary ammonium compounds, di-C12-18- 0.5 32 14.7 0.7 19, 48, 29
alkyldimethyl, nitrites 1.6 29.3 6.4 0.7 34, 32, 22
5 30.3 9.1 0.7 40, 22, 29
16 30.3 6.5 0.7 30, 24, 37
50 33 6.1 0.8 26, 36, 37
160 30.7 6.5 0.7 24, 37, 31
500 - - - - T, - T, - T
B[a]P 10 330 17.3 7.6 340, 310, 340
             
TA100 DMSO - 120.7 2.1 - 123, 119, 120
Quaternary ammonium compounds, di-C12-18- 0.5 88 4 0.7 84 M B, 88 M B, 92 M B
alkyldimethyl, nitrites 1.6 124 7.2 1 118, 122, 132
5 121.7 8.5 1 113 M, 122, 130
16 113.7 24.4 0.9 135, 87 M B, 119
50 113.7 31.1 0.9 139, 123, 79
160 101.7 8.4 0.8 92, 107, 106
500 - - - - T, - T, - T
AAN 5 1587 26.9 13.2 1556, 1604, 1601
             
TA1535 DMSO - 15 4.6 - 11, 14, 20
Quaternary ammonium compounds, di-C12-18- 0.5 13 2.6 0.9 16, 11, 12
alkyldimethyl, nitrites 1.6 11.3 2.3 0.8 10, 14, 10
5 17.3 4.6 1.2 20, 20, 12
16 12.7 7.6 0.8 6, 21, 11
50 19 5 1.3 19, 14, 24
160 13.7 1.5 0.9 12 S, 15 S, 14 S
500 - - - - T, - T, - T
  AAN 5 180.7 21.7 12 156, 197, 189
TA1537 DMSO - 11.7 2.1 - 10, 14, 11
Quaternary ammonium compounds, di-C12-18- 0.5 9.7 5.5 0.8 15, 4, 10
alkyldimethyl, nitrites 1.6 9.7 0.6 0.8 10, 10, 9
5 9 2.6 0.8 11, 10, 6
16 10.3 1.2 0.9 11, 11, 9
50 14.7 4 1.3 11, 14, 19
160 8 6.1 0.7 1 M P, 12 M P, 11 M P
500 0.7 1.2 0.1 0 M P, 0 M P, 2 M P
AAN 5 228.3 5.5 19.6 222, 232, 231
             
WP2uvrA DMSO - 31 2.6 - 29, 34, 30
Quaternary ammonium compounds, di-C12-18- 5 37.7 0.6 1.2 38, 37, 38
alkyldimethyl, nitrites 16 30.3 13.8 1 46, 20 M B, 25
50 29.7 3.2 1 26, 32, 31
160 25.7 0.6 0.8 26, 26, 25
500 10.7 5 0.3 10, 6, 16
1000 - - - - T, - T, - T
1600 - - - - T, - T, - T
AAN 15 240.7 43.7 7.8 219, 291, 212
         
Conclusions:
Quaternary ammonium compounds, di-C12 -18 -alkyldimethyl, nitrites, CAS No. 71487 -01 -9, EC No. 275 -532 -1, did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA) in the absence and in the presence of a rat liver metabolic activation system (S-9) in the OECD 471, Bacterial Reverse Phase Mutation study : Negative.
Executive summary:

A study was performed to evaluate the mutagenic potential of the test material (Quaternary ammonium compounds, di-C12 -18 -alkyldimethyl, nitrites, CAS No. 71487 -01 -9, EC No. 275 -532 -1)

The study was conducted according to OECD guideline 471 and according to GLP.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

These test conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines) and a toxic concentration, in the absence and in the presence of a rat liver metabolic activation system (S-9) in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA).

Under the conditions of the study it was concluded that Quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA).
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1992
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Basic information given in tabular form as part of a screening publication and a NTP technical report. No information regarding bacteriotoxicity provided.
Justification for type of information:
It is suggested that read-across data from sodium nitrite is considered appropriate in order to arrive at a conclusion in terms of any health effects that may be incurred from exposure to "nitrite" whichever cation it may be associated with.

In 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded and that the counter anion could be regarded as "any anionic species" then it would seem that to consider available toxicological data on sodium nitrite, in order to evaluate any health effects that may be incurred from exposure to the nitrite anion (NO2-), is justifiable.

Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
no guideline available
Principles of method if other than guideline:
According to Zeiger et al. 1992, Environ. Mol. Mutagen. 19 (suppl. 21), 2-141
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium
Species / strain / cell type:
other: Salmonella typhimurium TA98, TA100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9 mix
Test concentrations with justification for top dose:
0, 100, 333, 1000, 1666, 3333, 6666, and 10000 µg/plate
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Additional information on results:
Sodium nitrite was mutagenic in Salmonella typhimurium strain TA100, with and without Aroclor 1254-induced hamster and rat liver S9 enzymes; no mutagenicity was observed in strain TA98.
Conclusions:
Sodium nitrite was found to be mutagenic, with and without metabolic activation, in Salmonella typhimurium strain TA100 but non-mutagenic in the strain TA98.
Executive summary:

In a publication entitled "Salmonella mutagenicity tests: V. Results from the testing of 311 chemicals" ( Environ. Mol. Mutagen. 19 (Suppl. 21), 2-141., 1992; Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., and Mortelmans, K.) as a primary source, the in vitro mutagenic potential of sodium nitrite has been investigated.

This publication is also referenced in :-

Toxicology and Carcinogenesis Studies of Sodium Nitrite in F344/N Rats and B6C3f1 Mice (Drinking Water Studies), National Toxicology Program (NTP) Technical Report, (2001; NIH Publication No. 01-3954)

The Screenimg Information DataSet (SIDS) for sodium nitrite as a secondary source and in the disseminated REACH regiatration dossier for sodium nitrite.

The study was assigned a reliability score of 4 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997) since only basic information given in tabular form as part of a screening publication and a NTP technical report. No information regarding bacteriotoxicity provided.

Under the conditions of the study sodium nitrite was found to be mutagenic, with and without metabolic activation, in Salmonella typhimurium strain TA100 but non-mutagenic in the strain TA98.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Remarks:
Type of genotoxicity: gene mutation
Adequacy of study:
supporting study
Study period:
1984
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Basic information given. Non-guideline study with the following restrictions: No E. coli strains were tested, no positive controls were run, information regarding bacteriotoxicity of the test substance is not provided.
Justification for type of information:
It is suggested that read-across data from sodium nitrite is considered appropriate in order to arrive at a conclusion in terms of any health effects that may be incurred from exposure to "nitrite" whichever cation it may be associated with.

In 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded and that the counter anion could be regarded as "any anionic species" then it would seem that to consider available toxicological data on sodium nitrite, in order to evaluate any health effects that may be incurred from exposure to the nitrite anion (NO2-), is justifiable.

Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
no guideline available
Principles of method if other than guideline:
according to the method of Ames, McCann & Yamasaki (1975)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Sodium nitrite, purity 97%
Target gene:
various S. typhimurium strains
Species / strain / cell type:
other: Salmonella typhimurium TA1535, TA 1537, TA 92, TA 94, TA 98, TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9 mix
Test concentrations with justification for top dose:
1 - 10000 ug/plate (TA 1535, TA 100), 10 - 5000 ug/plate (TA 1537, TA 92, TA 94, TA 98)
Vehicle / solvent:
phosphate buffer
Details on test system and experimental conditions:
The liver microsome fraction (S-9) was prepared from the liver of Fischer rats prepared 5 days before with polychlorinated biphenyls (500 mg/kg body weight of Kanechlor KC-400 in olive oil, ip). The reaction mixture (S-9 mix) contained 5 mM-glucose 6-phosphate, 4 mM-NADPH, 4 mM-NADH, 33 mM-KCI, 8mM-MgCI2 100 mM-phosphate buffer (pH 7.4) and 3.75 ml S-9 (129 mg protein) in a total volume of 12.5 ml. Cells cultured ovemight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37°C before plating. Duplicate plates were used for each of six different concentrations of the sample. The number of revertant (his+) colonies was scored after incubation at 37°C for 2 days.
Evaluation criteria:
The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays (Yoshikawa, Nakadate, Watabe et al. 1980) were performed.
Key result
Species / strain:
other: Salmonella typhimurium TA1535, TA 1537, TA 92, TA 94, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Without S9 mix, there was a slight decrease in the number of revertants at doses above 5 mg/plate. 800 revertants/plate at 10 mg/plate in TA1535 with S-9 mix. 2800 revertants/plate at 10 mg/plate in TA1535 with S-9 mix and 2069 rev./plate without S-9 mix; 465 rev./plate at 10 mg/plate in TA100 with S-9 mix and 314 rev./plate without S-9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Salmonella typhimurium
Conclusions:
Sodium nitrite was found to be mutagenic, with and without metabolic activation, in Salmonella typhimurium strains TA1535, TA 1537, TA 92, TA 94, TA 98, TA 100.
Executive summary:

In a publication entitled "Primary mutagenicity screening of food additives currently used in Japan." ( Chem. Toxicol. 22, 623-636, 1984; Ishidate, M., Jr., Sofuni, T., Yoshikawa, K., Hayashi, M., Nohmi, T., Sawada, M., and Matsuoka, A..) as a primary source, the in vitro mutagenic potential of sodium nitrite has been investigated.

This publication is also referenced in :-

Toxicology and Carcinogenesis Studies of Sodium Nitrite in F344/N Rats and B6C3f1 Mice (Drinking Water Studies), National Toxicology Program (NTP) Technical Report, (2001; NIH Publication No. 01-3954)

The Screenimg Information DataSet (SIDS) for sodium nitrite as a secondary source and in the disseminated REACH regiatration dossier for sodium nitrite.

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997) since only basic information given. Non-guideline study with the following restrictions: No E. coli strains were tested, no positive controls were run, information regarding bacteriotoxicity of the test substance is not provided.

Under the conditions of the study sodium nitrite was found to be mutagenic, with and without metabolic activation, in Salmonella typhimurium strains TA1535, TA 1537, TA 92, TA 94, TA 98, TA 100.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Remarks:
Type of genotoxicity: chromosome aberration
Adequacy of study:
weight of evidence
Study period:
1977
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Basic information given. Non-guideline study with the following restrictions: Tested concentrations not indicated, no information regarding statistical evaluation provided.
Justification for type of information:
It is suggested that read-across data from sodium nitrite is considered appropriate in order to arrive at a conclusion in terms of any health effects that may be incurred from exposure to "nitrite" whichever cation it may be associated with.

In 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded and that the counter anion could be regarded as "any anionic species" then it would seem that to consider available toxicological data on sodium nitrite, in order to evaluate any health effects that may be incurred from exposure to the nitrite anion (NO2-), is justifiable.

Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
no guideline available
Principles of method if other than guideline:
Screening data on 134 chemicals
GLP compliance:
no
Type of assay:
other: in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Sodium nitrite
Target gene:
Chinese hamster fibroblast cell line (CHL)
Species / strain / cell type:
other: Chinese hamster fibroblast cell line (CHL)
Metabolic activation:
without
Metabolic activation system:
rat liver S-9 mix
Test concentrations with justification for top dose:
up to 1.0 mg/ml
Vehicle / solvent:
saline
Details on test system and experimental conditions:
A clonal sub-line of a Chinese hamster fibroblast cell line (CHL) was used.This cell line was originally established from the lung of a young adult by Dr. T. Utakoji, Cancer Institute, Tokyo. The karyotype consisted of 25 chromosomes.The cells had been maintained by 5-day passages and grown in a monolayer in petri dishes with Eagle's MEM (GIBCO F-11) supplemented with 10%calf serum. Their doubling time was estimated as 18.2 h at their exponential growth at 37°C in a 5% CO2 atmosphere.Growth inhibition tests were carried out on each sample before the chromosome tests were started. The 50% inhibition dose was estimated as follows.Several different doses of each agent were separately added to the 3-day-old cultures (about 6 X 103 cells/3-cm dish). The doses were prepared by a factor of 2 from the maximal dose, estimated from the data on LD50, which appeared in references for each agent. The cells in a monolayer were washed, fixed with 10% formalin solution, and then stained with 0.1% crystal violet solution for 3 min. After washing and drying each dish was placed under a photodensitometer, which was designed in the laboratory and is being manufactured by Olympus Co., Japan, to measure the color absorption values from which relative cell densities on the dishes were easily calculated. The color absorption values obtained by this apparatus reflected well the actual number of cells that survived at the bottom of each petri dish.
Evaluation criteria:
CHL cells commonly have less than 3.0% cells with chromosomal aberrations. Therefore, the final judgement given to all experimental groups was as follows. Negative (-) if less than 4 .9% of the aberration was detected even when doses of the agent were elevated to sub-lethal amounts, where almost no mitosis was observed; suspicious (±) if between 5.0 and 9.9%, and positive if between 10.0 and 19.9% (+), 20.0 and 49.9% (++) or more than 50.0% (+++). When no reasonable dose response was obtained, additional experiments with different doses were carried out to confirm its reproducibility.
Key result
Species / strain:
other: Chinese hamster fibroblast cell line (CHL)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
The maximally effective dose was 0.53 mg/ml. Among the changes observed were: chromatid gaps, chromatid or chromosomal breaks and translocations.The number of cells with chromosomal aberrations was recorded on 100 well-spread metaphases at the magnification of 700.
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Conclusions:
Sodium nitrite was found to be mutagenic, without metabolic activation, in an in vitro mammalian chromosome aberration test on Chinese hamster fibroblast cell line (CHL); [Chromosome tests with 134 compounds on Chinese hamster cells in vitro - A Screening for Chemical Carcinogenesis, Ishidate, M., Odashima, S., Mutation Research 48, 337 - 354, 1977].
Executive summary:

In a publication entitled "Chromosome tests with 134 compounds on Chinese hamster cells in vitro - A Screening for Chemical Carcinogenesis" (Ishidate, M., Odashima, S., Mutation Research 48, 337 - 354, 1977) as a primary source, the in vitro mutagenic potential of sodium nitrite has been investigated in an in vitro cytogenicity / chromosome aberration study in mammalian cells.

This publication is also referenced in :-

Toxicology and Carcinogenesis Studies of Sodium Nitrite in F344/N Rats and B6C3f1 Mice (Drinking Water Studies), National Toxicology Program (NTP) Technical Report, (2001; NIH Publication No. 01-3954)

The Screenimg Information DataSet (SIDS) for sodium nitrite as a secondary source and in the disseminated REACH regiatration dossier for sodium nitrite.

The study was assigned a reliability score of 4 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997) since only basic information given. Non-guideline study with the following restrictions : Tested concentrations not indicated, no information regarding statistical evaulation is provided.

Under the conditions of the study sodium nitrite was found to be mutagenic, without metabolic activation, in this in vitro mammalian chromosome aberration test on Chinese hamster fibroblast cell line (CHL).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
It is suggested that read-across data from sodium nitrite is considered appropriate in order to arrive at a conclusion in terms of any health effects that may be incurred from exposure to "nitrite" whichever cation it may be associated with.

In 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded and that the counter anion could be regarded as "any anionic species" then it would seem that to consider available toxicological data on sodium nitrite, in order to evaluate any health effects that may be incurred from exposure to the nitrite anion (NO2-), is justifiable.

Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
no guideline available
Principles of method if other than guideline:
The purpose of this investigation was to examine the direct mutagenic and other biological effects of nitrite on mammalian cells.
GLP compliance:
no
Type of assay:
other: mammalian cell gene mutation assay
Specific details on test material used for the study:
Sodium nitrite
Target gene:
FM3A cell line from C3H mammary carcinoma
Species / strain / cell type:
other: FM3A cell line from C3H mammary carcinoma
Metabolic activation:
without
Metabolic activation system:
No data
Test concentrations with justification for top dose:
69-690 ug/ml (1-10 mMol)
Vehicle / solvent:
Eagle's minimal essential medium (MEM)
Details on test system and experimental conditions:
FM3A cells, a C3H mammary carcinoma cell line, were grown in suspension in Eagle's minimal essential medium ( MEM), containing 10% fetal bovine serum and antibiotics. Sodium nitrite was the product of Wako Pure Chemical Industries, Ltd . It was dissolved in MEM and diluted in the medium and sterilized through membrane filter before use.After the application of sodium nitrite and the treatment with 10-7 M colcemid for 2 h just before fixation, the cells were applied with 0 .9%o sodium citrate for 15 min, fixed and dehydrated with a mixture of inethanol and acetic acid and then spread on slides. Staining was done with Giemsa solution.
Key result
Species / strain:
other: FM3A cell line from C3H mammary carcinoma
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
The ratio of the NaNO2/control mutation frequencies was 3.9 at 69 ug/ml, 6.2 at 214 ug/ml and 49 at 690 ug/ml, denoting dose-dependent increase of the mutation frequency.
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Conclusions:
Sodium nitrite was shown to illicit a positive mutagenic result in an in vitro gene mutation study in mammalian cells (Mutagenic effect of sodium nitrite on cultured mouse cells, Kodama, F., Umeda, M., Tsutsui, T., Mutation Research 40, 119 - 124, 1976)
Executive summary:

In a publication entitled " Mutagenic effect of sodium nitrite on cultured mouse cells" ( Kodama, F., Umeda, M., Tsutsui, T., Mutation Research 40, 119 - 124, 1976) as a primary source, the in vitro mutagenic potential of sodium nitrite has been investigated in an in vitro gene mutation study in mammalian cells.

This publication is also referenced in :-

Toxicology and Carcinogenesis Studies of Sodium Nitrite in F344/N Rats and B6C3f1 Mice (Drinking Water Studies), National Toxicology Program (NTP) Technical Report, (2001; NIH Publication No. 01-3954)

The Screenimg Information DataSet (SIDS) for sodium nitrite as a secondary source and in the disseminated REACH regiatration dossier for sodium nitrite.

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997) since only basic information given.

Non-guideline study with the following restrictions: no information provided regarding cytotoxicity, choice of concentrations, replicates, and statistical evaluation.

Under the conditions of the study sodium nitrite was shown to illicit a positive mutagenic result in this in vitro gene mutation study in mammalian cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

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