1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with C18-unsatd. fatty acids, Me sulfates (salts)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in all tests conducted:
- Ames test with S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E coli WP2 uvrA (met. act.: with and without) (OECD TG 471 and GLP); S. typhimurium strains: tested up to cytotoxicity; E. coli WP 2 uvrA cytotoxicity: no
- Mammalian cell gene mutation assay with Mouse Lymphoma (L5178Y) cells (met. act.: with and without) (OECD Guideline 476 and GLP); tested up to cytotoxic concentrations; read-across from MDEA-Esterquat C16-18 and C18 unsatd.
- In vitro mammalian chromosome aberration test with Chinese Hamster Lung (CHL) cells (met. act.: with and without) (OECD Guideline 473 and GLP); cytotoxicity: no; read-across from MDEA-Esterquat C16-18 and C18 unsatd.

Link to relevant study records

Reference

Reference 1

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological and ecotoxicological properties because they share structural similarities with common functional groups: quaternary amines, esters, and fatty acid chains varying in their length and degree of (un)saturation. Moreover, the fatty acid chains are chemically simple structures which have no structural alerts for toxicity, and which are closely related to substances of known low toxicity (i.e. stearic acid, oleic acid, linoleic acid, linolenic acid). Furthermore, the substances can be expected to have comparable breakdown products (MDEA or MDIPA and long chain fatty acids).

This read-across hypothesis corresponds to scenario 2 - different compounds have qualitatively and quantitatively the same type of effects - of the read-across assessment framework i.e. properties of the target substance MDIPA-Esterquat C18 unsatd. are predicted to be similar to those of the source substance MDEA-Esterquat C16-18 and C18 unsatd.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See justification for read-across attached to chapter 13 of this IUCLID file.

3. ANALOGUE APPROACH JUSTIFICATION
See justification for read-across attached to chapter 13 of this IUCLID file.

4. DATA MATRIX
See justification for read-across attached to chapter 13 of this IUCLID file.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK)

Metabolic activation:

with and without

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

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological and ecotoxicological properties because they share structural similarities with common functional groups: quaternary amines, esters, and fatty acid chains varying in their length and degree of (un)saturation. Moreover, the fatty acid chains are chemically simple structures which have no structural alerts for toxicity, and which are closely related to substances of known low toxicity (i.e. stearic acid, oleic acid, linoleic acid, linolenic acid). Furthermore, the substances can be expected to have comparable breakdown products (MDEA or MDIPA and long chain fatty acids).

This read-across hypothesis corresponds to scenario 2 - different compounds have qualitatively and quantitatively the same type of effects - of the read-across assessment framework i.e. properties of the target substance MDIPA-Esterquat C18 unsatd. are predicted to be similar to those of the source substance MDEA-Esterquat C16-18 and C18 unsatd.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See justification for read-across attached to chapter 13 of this IUCLID file.

3. ANALOGUE APPROACH JUSTIFICATION
See justification for read-across attached to chapter 13 of this IUCLID file.

4. DATA MATRIX
See justification for read-across attached to chapter 13 of this IUCLID file.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Type of assay:

in vitro mammalian chromosome aberration test

Metabolic activation:

with and without

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

N/A

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

N/A

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative with and without metabolic activation

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-01-10 to 2011-02-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material: 1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C18 unsatd., Me-sulfates (salts)
- Name of test material (as cited in study report): HH-2010-324
- Physical state: liquid
- Analytical purity: 100%

Target gene:

Histidine locus (Salmonella typhimurium strains) and tryptophan locus (E. coli strain)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: Tester strains TA98 and TA1537 are reverted by frameshift mutagens. Tester strain TA1535 is reverted by basepair substitution mutagens. Tester strain TA100 is reverted by frameshift and basepair substitution mutagens.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

First experiment: 0 (control), 62, 185, 556, 1667 and 5000 µg/plate
Second experiment: 0 (control), 22, 67, 200, 600 and 1800 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: not given

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 (test substance, positive controls), 6 (negative controls) in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies attended by a clearing of the background lawn

Evaluation criteria:

A test substance producing no biologically relevant positive response in any one of the bacterial strains tested is considered to be non-mutagenic in this system.
A biologically relevant response is described as folIows:
If the number of revertants is at least twice the spontaneous reversion rate for TA 1535, TA 98, TA 100 or WP2 uvrA (or three times for TA 1537) and/or if there is a concentration related increasing number of revertants over the range tested.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle 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

Vehicle 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

Vehicle 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

Vehicle 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

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Precipitation: Precipitation was observed when the test substance was pipetted into the top agar and later on the agar plates beginning at 556 µg/plate with and without metabolic activation

COMPARISON WITH HISTORICAL CONTROL DATA:
All mean numbers of revertant colonies on negative control plates fell within the ranges of the historical control data and were significantly elevated by positive control treatments.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects were noted starting at 1667 µg/plate in strains TA 1535 without metabolic activation, TA 1537, TA 98 and TA 100 with and without metabolic activation and at 5000 µg/plate in TA 1535 with metabolic activation; no cytotoxic effects were seen in E. coli WP 2 uvrA.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

MDIPA Esterquat C18 unsatd. was evaluated in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of rat liver S9 mix. Under the conditions of the study, the test substance was negative for mutagenic potential.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997) and EU method B.13/14 (30 May 2008), strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium and Escherichia coli WP2 uvrA were exposed to MDIPA Esterquat C18 unsatd.

(100% a.i.) in acetone at concentrations of 0 (control), 62, 185, 556, 1667 and 5000 µg/plate in the first experiment and 0 (control), 22, 67, 200, 600 and 1800 µg/plate in the second experiment in the presence and absence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method.

MDIPA Esterquat C18 unsatd. was tested up to cytotoxic concentrations. Cytotoxic effects were noted starting at 1667 µg/plate in strains TA 1535 without metabolic activation, TA 1537, TA 98 and TA 100 with and without metabolic activation and at 5000 µg/plate in TA 1535 with metabolic activation; no cytotoxic effects were seen in E. coli WP 2 uvrA.

Precipitation was observed when the test substance was pipetted into the top agar and later on the agar plates beginning at 556 µg/plate with and without metabolic activation.

The positive controls induced the appropriate responses in the corresponding strains.The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of induced mutant colonies over background.

Under the conditions of the study, the test substance was negative for mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For the assessment of the mutagenic potential of MDIPA-Esterquat C18 unsatd. a bacterial reverse mutation assay is available. A mammalian cell gene mutation assay, a mammalian cell cytogenetics assay as well as a mouse bone marrow micronucleus assay is available for the source substance MDEA-Esterquat C16-18 and C18 unsatd. A justification for read-across is attached to Iuclid section 13.

In vitro tests

Reverse gene mutation assays in bacteria

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997) and EU method B.13/14 (30 May 2008), strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium and Escherichia coli WP2 uvrA were exposed to the target substance MDIPA Esterquat C18 unsatd. (100% a.i.) in acetone at concentrations of 0 (control), 62, 185, 556, 1667 and 5000 µg/plate in the first experiment and 0 (control), 22, 67, 200, 600 and 1800 µg/plate in the second experiment in the presence and absence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method.

MDIPA Esterquat C18 unsatd. was tested up to cytotoxic concentrations. Cytotoxic effects were noted starting at 1667 µg/plate in strains TA 1535 without metabolic activation, TA 1537, TA 98 and TA 100 with and without metabolic activation and at 5000 µg/plate in TA 1535 with metabolic activation; no cytotoxic effects were seen in E. coli WP 2 uvrA. Precipitation was observed when the test substance was pipetted into the top agar and later on the agar plates beginning at 556 µg/plate with and without metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data. There was no evidence of induced mutant colonies over background.

Under the conditions of the study, MDIPA Esterquat C18 unsatd. was negative for mutagenic potential.

 

For the structurally similar source substance MDEA-Esterquat C16-18 and C18 unsatd., a reverse bacterial gene mutation assay (Ames-Test, plate incorporation assay) according to OECD Guideline 471 (February 1998) was negative up to the limit concentration of 5000 µg/plate with and without mammalian metabolic activation (Aroclor 1254 induced rat liver S9-mix) in S. Typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA (pKM101). MDEA-Esterquat C16-18 and C18 unsatd . was tested at concentrations of 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate, concentrations of 1500 and 5000 µg/plate were cytotoxic. Precipitation was observed beginning at 1500 or at 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in any of the tester strains in the presence or absence of mammalian metabolic activation. Under the conditions of the study, MDEA-Esterquat C16-18 and C18 unsatd. was negative for mutagenic potential.

 

Also the second source substance MDIPA-Esterquat C16-18 and C18 unsatd. was tested in a reverse gene mutation assay in bacteria according to OECD guideline 471 using strains TA 1535, TA 1537, TA 98, TA 100 of S. typhimurium and E. coli WP2. The test item was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

 

Mammalian cell gene mutation assay

Additional data are for the source substance MDEA-Esterquat C16-18 and C18 unsatd. is available from a mammalian cell gene mutation assay (thymidine kinase locus) comparable to OECD Guideline 476, performed with mouse lymphoma L 5178Y cells. Cells cultured in vitro were exposed to MDEA-Esterquat C16-18 and C18 unsatd. at concentration up to 150 µg/mL in the absence and up to 550 µg/mL in the presence of mammalian metabolic activation (S9- mix of Aroclor 1254 induced rat liver).

Substantial toxicity with a suspension growth of ≤ 50 % was observed at ≥ 50 µg/mL without metabolic activation and at ≥ 150 µg/mL with metabolic activation.

In the first experiment the concentration range did not cover cytotoxicity in the test with metabolic activation. Due to unacceptable high solvent controls of the test with metabolic activation in the confirmatory assay a third independent assay was performed.

No treated cultures with or without metabolic activation with ≥ 10 % total growth exhibited ≥ 100 induced mutants per 1E06 clonable cells over background level, the limit value for a positive response. No treated cultures without metabolic activation with ≥ 10 % total growth exhibited ≥ 55 to 99 induced mutants per 1E06 clonable cells over background level, the limit values for equivocal response.

Three cultures with metabolic activation with ≥ 10 % total growth showed induced mutant frequency of ≥ 55 induced mutants per 1E06 clonable cells over background at a concentrations of 40, 60 and 75 µg/mL in the first assay (63, 71 an 56 induced mutants per 1E06 clonable cells over background, respectively). A mutant number in the range of negative results was observed at the intermediate concentration of 50 µg/mL, (41 induced mutants per 1E06 clonable cells over background). A dose-response trend was not observed. 

In the third assay much higher concentrations from 110 to 550 µg/mL were tested, due to the missing cytotoxicity in the first assay. At the intermediate concentration of 300 µg/mL, a mutant number equivalent to the lower bound for equivocal results of 55 induced mutants per 1E06 clonable cells over background was observed. A dose-response trend was not observed.

The positive controls did induce the appropriate response. 

The results of the L 5178Y/ TK Mouse Lymphoma Mutagenesis Assay indicate that, under the conditions of this study the MDEA-Esterquat C16-18 and C18 unsatd. did not cause a positive response in the non-activated and S9-activated systems and was concluded to be negative.

 

Mammalian cell cytogenetics assay

In a mammalian cell cytogenetics assay, chromosome aberration test comparable to OECD Guideline 473 (21 July 1997), CHO cell cultures were exposed to the source substance MDEA-Esterquat C16-18 and C18 unsatd. (99.8 % a.i.), at concentrations of 25, 50, 100, 200, 225, 250 and 275µg/mL in the non-activated 4-hour treatment group and of 25, 50, 100, 125, 150, 175 and 200 µg/mL in the non-activated 20-hour treatment group. 

Aroclor 1254-induced rat liver S9-mix was used for metabolic activation at test substance concentrations of 25, 50, 100, 200, 225, 250, 275 and 300 µg/mL in the activated 4-hour treatment group.

Substantial toxicity (at least 50% reduction in cell growth, relative to the solvent control), was observed at dose levels ≥ 200μg/mL in the non-activated 4-hour treatment group, at dose levels ≥ 225μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 125μg/mL in the non-activated 20-hour treatment group.

Selection of doses for microscopic analysis was based on toxicity including the lowest dose with at least 50% reduction in cell growth relative to solvent control in all three treatment groups. The following doses were selected for microscopic evaluation:

non-activated 4-hour treatment group: 50, 100 and 200μg/mL

S9-activated 4-hour exposure group: 100, 200 and 225μg/mL

non-activated 20-hour treatment group: 50, 100 and 125μg/mL

Positive controls induced the appropriate response.

MDEA-Esterquat C16-18 and C18 unsatd. was concluded to be negative for the induction of structural and numerical chromosome aberrations in both the presence and absence of metabolic activation in Chinese hamster ovary (CHO) cells.

 

IN VIVO TEST

Mouse bone marrow micronucleus assay

In a NMRI BR mouse bone marrow micronucleus assay according to OECD guideline 474 (adopted July 21, 1997) and EU method B.12 (31 May 2008) 5 animals/sex/dose were treated by intraperitoneal injection with the source substance MDEA-Esterquat C16-18 and C18 unsatd. at doses of 0, 500, 1000 and 2000 mg/kg bw.  Bone marrow cells were harvested at 24 h for all dose levels and additionally at 48 h at 2000 mg/kg bw post-treatment.  The vehicle was corn oil.

Hunched posture, rough coat and lethargy were observed at 1000 and 2000 mg/kg bw; hunched posture and rough coat were also present at 500 mg/kg bw. MDEA-Esterquat C16-18 and C18 unsatd. was tested up to the maximum recommended dose in accordance with current regulatory guidelines. No decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis. However the route of exposure was chosen to maximise the chance of the test substance reaching the target tissue. The positive control induced the appropriate response.There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

 

Based on the available reliable, relevant and adequate data, there was no evidence of genotoxicity for MDIPA-Esterquat C18 unsatd. There are no data gaps for the endpoint genotoxicity. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

 

 

Conclusion

The structural similarities between the source and the target substances and the similarities in their breakdown products presented above support the read-across hypothesis. Adequate and reliable scientific information indicates that the source and target substances and their subsequent degradation products have similar toxicity profiles as demonstrated in detail in the general justification for read-across.

The negative results from the bacterial reverse mutation assay, which is available for the source substances and the target substance, justify this read-across approach.

Further support is given by the lacking skin sensitisation potential for both, source and target substances. The endpoint sensitisation is – similar to the endpoint genotoxicity – based on covalent binding of the substance itself or reactive metabolites to cellular macromolecules as rate determining step. The consistency across the endpoints increases the confidence in the conclusion that there is no concern for reactive metabolites. 

 

The negative outcome of the complete testing battery of in vitro and in vivo genotoxicity tests for MDEA-Esterquat C16-18 and C18 unsatd. is considered to be relevant also for the target MDIPA-Esterquat C18 unsatd. No classification for genotoxic properties is required. 

 

Justification for classification or non-classification

There was no evidence for any genotoxic intrinsic properties in the Ames-Test, mouse lymphoma assay and chromosome aberration study and MDIPA Esterquat C18 unsatd. is therefore considered to be non-genotoxic. MDIPA Esterquat C18 unsatd. is not to be classified as genotoxic according to Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008. No labelling is required.