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Administrative data

Key value for chemical safety assessment

Additional information

Not all data on genetic toxicity are available for the target substance MDEA Esterquat C18 satd.. For the assessment of effects of MDEA Esterquat C18 satd. on mutagenicity results from the following studies are taken into consideration:

- an OECD 471 reverse gene mutation assay in bacteria and an OECD 476 mammalian gene mutation assay of source substance MDEA Esterquat C16-18 and C18 unsatd.

- an OECD 473in vitro mammalian chromosome aberrationof the target substanceMDEA Esterquat C18 satd.

- an additional OECD 473473in vitro mammalian chromosome aberrationof thesource substanceMDEA-Esterquat C16-18 and C18 unsatd. for the justification for read across for the endpoint genetic toxicity

- an OECD 474 in vivo micronucleus test of thesource substanceMDEA-Esterquat C16-18 and C18 unsatd.

 

In vitro tests

Reverse gene mutation assays in bacteria

A reverse bacterial gene mutation assay (Ames-Test, plate incorporation assay) with the structurally similar source substance MDEA-Esterquat C16-18 and C18 unsatd. 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.

Mammalian cell cytogenetics assay

In an in vitro mammalian chromosome aberration assay according to EU Method B.10 (Mutagenicity – In Vitro Mammalian Aberration Test) dated May, 30, 2008 and OECD Guideline 473 (In Vitro Mammalian chromosome Aberration Test) adopted 21stJuly, 1997 V79 Chinese Hamster cells cultured in vitro were exposed to MDEA Esterquat C18 satd. (98.5%) prepared in water in the presence and absence of mammalian metabolic activation with S9 homogenate.

The chromosomes were prepared 20 h after start of treatment with the test item. Two parallel cultures were set up. 100 metaphases per culture were scored for structural chromosomal aberrations.

 

The following concentrations were evaluated for microscopic analysis:

 

Experiment I:

Without metabolic activation (4h treatment): 1000, 2000 and 2500 µg/mL

With metabolic activation (4h treatment): 2500, 3500 and 5000 µg/mL

 

Experiment II:

Without metabolic activation (20h treatment): 125, 250 and 500 µg/mL

With metabolic activation (4h treatment): 2000, 2600, 3800 and 5000 µg/mL

 

There was no evidence of a biologically relevant increase of the aberration rates or increase in the frequencies of polyploid cells over background in both experiments with and without metabolic activation.The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

The positive controls EMS (Ethylmethanesulfonate, 400 and 600 µg/mL) and CPA (Cyclophosphamide, 0.83 µg/mL) induced distinct increases of structural chromosomal aberrations.

 

A similar result was obtained with structurally similar source substance MDEA-Esterquat C16-18 and C18 unsatd.:

In a mammalian cell cytogenetics assay, chromosome aberration test comparable to OECD Guideline 473 (21 July 1997),CHO cell cultures were exposed to MDEA-Esterquat C16-18 and C18 unsatd. (99.8 % a.i.), at concentrations of 25, 50, 100, 200, 225, 250 and 275 µg/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 ≥ 200 μg/mL in the non-activated 4-hour treatment group, at dose levels ≥ 225 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 125 μg/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 200 μg/mL

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

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

Mammalian cell gene mutation assay

Additional data of the structurally similar source substance MDEA-Esterquat C16-18 and C18 unsatd. are 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 did not cause a positive response in the non-activated and S9-activated systems and was concluded to be negative.

 

In vivo test

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 structurally similar 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.

Justification for read-across

For details on substance identity, toxicokinetics and detailed toxicological profiles, please refer also to the general justification for read-across given in chapter 5 of the CSR and attached as pdf document to section 7 of the IUCLID file.

 

a. Structural similarity and functional groups

The target substance, MDEA Esterquat C18 satd., consists of an amine backbone (MDEA = Methyldiethanol amine) esterified with the long chain fatty acid stearic acid (C18 satd.; IV (iodine value) < 1). The main reaction product is the dialkylester compound, next to that small amounts of the monoalkylester may be formed. The amine function is quaternised with two methyl groups. The counter ion is Chloride.

The source substance, MDEA EsterquatC16-18 and C18 unsatd., consists of the same amine backbone (MDEA = Methyldiethanol amine) but esterified with a mixture of the long chain fatty acids C16, C18 and C18 unsaturated (IV < 25). The main reaction product is the dialkylester compound, next to that small amounts of the monoalkylester may be formed. The amine function is quaternised with two methyl groups. The counter ion is Chloride.

The source and the target substance share structural similarities with common functional groups (quaternary amines, esters, and fatty acid chains with comparable length and degree of saturation).

b. Common breakdown products

The metabolism expected to occur is hydrolysis of the ester-bond by esterases. However, the rate of hydrolysis is assumed to be low. The fraction of metabolised molecules would result in free fatty acids and Dimethyl-DEA (DEA = Diethanolamine). The carboxylic acids are further degraded by the mitochondrial beta-oxidation process (for details see common text books on biochemistry). The fatty acids enter normal metabolic pathways and are therefore indistinguishable from fatty acids from other sources including diet. The quaternary ammonium ions are not expected to be further metabolised, but excreted unchanged via the urine. 

c. Differences

The differences in fatty acid chain length (higher percentage of C16 in the source substance MDEA Esterquat C16 -18 and C18 unsatd.) and degree of saturation (higher degree of unsaturation of the C18 fatty acid chains in the source substance substance MDEA Esterquat C16-18 and C18 unsatd.) could be relevant for local toxicity (skin and eye irritation) but arebut are not considered to be of relevance for genetic toxicity.

Genetic toxicity data of the target and source substance

 

Comparison of the acute toxicity data of MDEA Esterquat C18 satd. and MDEA-Esterquat C16-18 and C18 unsatd.

 

Source substance

Target substance

 

Endpoints

 

MDEA-Esterquat C16-18 and C18 unsatd.

 

MDEA Esterquat C18 satd.

Genotoxicity in vitro

 

 

 

 

 

 

 

 

 

 

 

 

 

 

accordingto OECD TG 471 (Ames test)
RL 1, GLP,
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 (met. act.: with and without)

 

negative for all strains (with and without metabolic activation)

 

Doses:

S. typhimurium/E.coli (w/o and w met act.): 15, 50, 150, 500, 1500 and 5000 µg/plate

 

cytotoxicity: yes

 

non-mutagenic

 

Read-across from the source substance

equivalent to OECD TG 476 (Mammalian cell gene mutation, MLY), RL 2, GLP

negative for mouse lymphoma L5178Y cells (with and without metabolic activation)

 

Doses:

5, 10,

,22.5, 25, 27.5, 30, 32.5, 35, 40, 50, 60, 75, 90, 100, 110, 120, 130, 140 and 150 µg/mL (non-activated system) and 25, 27.5, 30, 32.5, 35, 40, 50, 60, 75, 90, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500 and 550 µg/mL (S9-activated system).

 

cytotoxicity: yes

 

non-mutagenic

Read-across from the source substance

 

according to OECD TG 473 (In vitro mammalian chromosome aberration), RL 1, GLP

negative for Chinese hamster Ovary (CHO) cells (with and without metabolic activation)

 

cytotoxicity: yes

 

non-mutagenic

according to OECD TG 473 (In vitro mammalian chromosome aberration), RL 1, GLP

negative for Chinese hamster V79 cells (with and without metabolic activation)

 

cytotoxicity: yes

 

non-mutagenic

Genotoxicity in vivo

according to OECD TG 474 (Mammalien Erythrocyte Micronucleus Test), RL 1, GLP

no induction of an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time

 

Doses:

0, 500, 1000 and 2000 mg/kg bw

 

Toxicity: no effects

 

non-mutagenic

Read-across from the source substance

 

 

The source substance MDEA-Esterquat C16-18 and C18 unsatd. and the target substance MDEA Esterquat C18 satd. showed the same negative results in the In vitro mammalian chromosome aberration assay (not mutagenic up to cytotoxic concentrations). There was no evidence of a biologically relevant increase of the aberration rates or increase in the frequencies of polyploid cells over background in both assays with and without metabolic activation. These results further justify the read-across approach by extrapolation from the source substances for the mammalian gene mutation assay and the bacterial reverse mutation assay. Additional safety gives the negative in vivo micronucleus test with the source substance MDEA-Esterquat C16-18 and C18 unsatd.

 

 

Quality of the experimental data of the analogues:

The target substance MDEA Esterquat C18 satd. has been tested in a reliable GLP-compliant study equivalent to OECD 473. The source substance MDEA-Esterquat C16-18 and C18 unsatd. has been tested in reliable studies according or similar to OECD TG 471, OECD TG 476, OECD TG 473 and OECD TG 474. All tests have been conducted according to GLP criteria.

Therefore this data have no uncertainties and can be used in an analogue approach.

The available data from the source chemical are sufficiently reliable to justify the read-across approach.

 

Classification and labelling (Human Health)

The source substance MDEA-Esterquat C16-18 and C18 unsatd. is not classified for any human health effects similar to the target substance MDEA Esterquat C18 satd. which is also not classified regarding human health hazards.

 

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 theIn vitro mammalian chromosome aberrationassay, which is available for the source substances and the target substance, justify this read-across approach.

 

The negative outcome of the complete testing battery of in vitro genotoxicity tests for MDEA-Esterquat C16-18 and C18 unsatd. is considered to be relevant also for the target substance MDEA Esterquat C18 satd..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.


Justification for selection of genetic toxicity endpoint
No single key study has been selected since all available studies were negative.Conclusion based on the following assays: Bacterial reverse mutation assay (Ames test); Mammalian cell gene mutation assay; In vitro mammalian chromosome aberration test and in vivo micronucleus test

Short 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, GLP); tested up to cytotoxic concentrations; read-across from source substance MDEA-Esterquat C16-18 and C18 unsatd.
- Mammalian cell gene mutation assay with Mouse Lymphoma (L5178Y) cells (met. act.: with and without) (OECD Guideline 476, GLP); tested up to cytotoxic concentrations; read-across from source substance MDEA-Esterquat C16-18 and C18 unsatd.
- In vitro mammalian chromosome aberration test with Chinese Hamster V79 cells (met. act.: with and without) (OECD Guideline 473, GLP); cytotoxicity: yes
- in vivo bone marrow micronucleus assay in NMRI BR mouse (OECD guideline 474, GLP); tested up to maximum recommended dose of 2000 mg/kg bw, intraperitoneal; no toxic effects; read-across from source substance MDEA-Esterquat C16-18 and C18 unsatd.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

There was no evidence for any genotoxic intrinsic properties in the chromosome aberration study conducted with the substance itself and in a mouse lymphoma assay, a Ames test and a in vivo micronucleus study conducted with a structurally closely related substance.MDEA Esterquat C18 satd. is therefore considered to be non-genotoxic.MDEA Esterquat C18 satd. 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.