(carboxymethyl)dimethyl-3-[(1-oxododecyl)amino]propylammonium hydroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro data from several gene mutation studies in bacteria (Ames Tests) on C8 -18 and C18 unsatd. AAPB, one in vitro gene mutation study in mammalian cells (L5178Y/ TK Mouse Lymphoma assay) performed on C8-18 AAPB and one cytogenicity study (in vivo Mammalian Erythrocyte Micronucleus Test) on C8 -18 and C18 unsatd. AAPB are available. All tests were consistently negative. There is no evidence for a genotoxic potential of AAPBs.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
see "General Justification for Read-Across" attached to IUCLID section 13

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Mutual read across from the AAPBs to one another is justified:

a) Based on the information given in section 1, it can be concluded that all AAPBs mentioned above are similar in structure, since they are manufactured from similar resp. identical precursors under similar conditions and all contain the same functional groups. Thus a common mode of action can be assumed.
b) The content of minor constituents in all products are comparable and differ to an irrelevant amount.
c) The only deviation within this group of substances is a minor variety in their fatty acid moiety, which is not expected to have a relevant impact on intrinsic toxic or ecotoxic activity and environmental fate. Potential minor impact on specific endpoints will be discussed in the specific endpoint sections.

The read-across hypothesis is based on structural similarity of target and source substances. Based on the available experimental data, including key physico-chemical properties and data from toxicokinetic, acute toxicity, irritation, sensitisation, genotoxicity and repeated dose toxicity studies, the read-across strategy is supported by a quite similar toxicological profile of all five substances.
The respective data are summarised in the data matrix; robust study summaries are included in the Technical Dossier in the respective sections.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see "General Justification for Read-Across" attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see "General Justification for Read-Across" attached to IUCLID section 13

4. DATA MATRIX
see "General Justification for Read-Across" attached to IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 100, TA 1537, TA 1538 and TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

negative with and without metabolic activation

Reference 2

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
see "General Justification for Read-Across" attached to IUCLID section 13

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Mutual read across from the AAPBs to one another is justified:

a) Based on the information given in section 1, it can be concluded that all AAPBs mentioned above are similar in structure, since they are manufactured from similar resp. identical precursors under similar conditions and all contain the same functional groups. Thus a common mode of action can be assumed.
b) The content of minor constituents in all products are comparable and differ to an irrelevant amount.
c) The only deviation within this group of substances is a minor variety in their fatty acid moiety, which is not expected to have a relevant impact on intrinsic toxic or ecotoxic activity and environmental fate. Potential minor impact on specific endpoints will be discussed in the specific endpoint sections.

The read-across hypothesis is based on structural similarity of target and source substances. Based on the available experimental data, including key physico-chemical properties and data from toxicokinetic, acute toxicity, irritation, sensitisation, genotoxicity and repeated dose toxicity studies, the read-across strategy is supported by a quite similar toxicological profile of all five substances.
The respective data are summarised in the data matrix; robust study summaries are included in the Technical Dossier in the respective sections.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see "General Justification for Read-Across" attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see "General Justification for Read-Across" attached to IUCLID section 13

4. DATA MATRIX
see "General Justification for Read-Across" attached to IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase Locus

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

Positive controls validity:

valid

Conclusions:

The test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A cytogenicity study (in vivo Mammalian Erythrocyte Micronucleus Test) on C8 -18 and C18 unsatd. AAPB is available, which showed no genotoxic potential.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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
see "General Justification for Read-Across" attached to IUCLID section 13

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Mutual read across from the AAPBs to one another is justified:

a) Based on the information given in section 1, it can be concluded that all AAPBs mentioned above are similar in structure, since they are manufactured from similar resp. identical precursors under similar conditions and all contain the same functional groups. Thus a common mode of action can be assumed.
b) The content of minor constituents in all products are comparable and differ to an irrelevant amount.
c) The only deviation within this group of substances is a minor variety in their fatty acid moiety, which is not expected to have a relevant impact on intrinsic toxic or ecotoxic activity and environmental fate. Potential minor impact on specific endpoints will be discussed in the specific endpoint sections.

The read-across hypothesis is based on structural similarity of target and source substances. Based on the available experimental data, including key physico-chemical properties and data from toxicokinetic, acute toxicity, irritation, sensitisation, genotoxicity and repeated dose toxicity studies, the read-across strategy is supported by a quite similar toxicological profile of all five substances.
The respective data are summarised in the data matrix; robust study summaries are included in the Technical Dossier in the respective sections.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see "General Justification for Read-Across" attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see "General Justification for Read-Across" attached to IUCLID section 13

4. DATA MATRIX
see "General Justification for Read-Across" attached to IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

in preliminary dose finding study lethal at doses >/= 1000 mg/kg bw

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test substance did not induce clastogenic effects in this in vivo cytogenicity study on mice at dose levels of 20 and 200 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No experimental data on genetic toxicity are available for C12 AAPB. However, reliable, relevant and adequate data on the genetic toxicity of AAPBs is available from several in vitro gene mutation studies in bacteria (Ames Tests) conducted with the closely related substance C8-18 and C18 unsatd. AAPB (Coco AAPB), one in vitro gene mutation study in mammalian cells (L5178Y/ TK Mouse Lymphoma assay) conducted with the closely related substance C8-18 AAPB and onein vivocytogenicity study (in vivoMammalian Erythrocyte Micronucleus Test) conducted with the closely related substance C8-18 and C18 unsatd. AAPB.

All tests were consistently negative. There is no evidence for a genotoxic potential of AAPBs.

A justification for read-across is given below.

 

In vitro data

In the key in vitro reverse gene mutation assay in bacteria performed according to EU Method B.14 (Version Commission Directive 92/69/EEC), strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 ofSalmonella typhimuriumwere exposed to C8-18 and C18 unsatd. AAPB. Test was performed with concentrations up to and including cytotoxic concentrations in the absence and the presence of mammalian metabolic activation.

No evidence of biologically significant mutagenic activity of the test item was found in the presence and absence of metabolic activation, up to and including its cytotoxic limit. The positive controls induced the appropriate responses in the corresponding strains and activity of metabolizing system was confirmed.

There was no evidence of induced mutant colonies over background.

The adopted OECD TG 471 (1997) requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, AAPB is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 and EU Method B.13/14 (Version Commission Directive 92/69/EEC) without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of AAPB in this bacterial test system.

Consistently negative results were also seen in several further supporting in vitro gene mutation assays in bacteria performed on Coco AAPB.

 

In a mammalian cell gene mutation assay according to OECD Guideline 476 (1997) and EU Method B.17 (2008), the potential of C8 -18 AAPB to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y was tested.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix: 2.4; 4.9; 9.8; 19.5; and 39.0 µg/mL

with S9 mix: 4.9; 9.8; 19.5; 39.0; and 78.0 µg/mL

Experiment II:

without S9 mix: 10; 20; 40; 50; and 60 µg/mL

with S9 mix: 40: 80; 100; 110; and 120 µg/mL

Relevant cytotoxic effects indicated by a relative total growth of less than 50% in both parallel cultures were observed in the absence of metabolic activation at 39 µg/mL in experiment I following 4 hour treatment and at 40 µg/mL and above in experiment II following 24 hours treatment. In the presence of metabolic activation toxic effects as described above occurred at 100 µg/mL and above in experiment II. No reproducible cytotoxic effects were noted in the first experiment with metabolic activation. The recommended toxic range of approximately 10-20 % relative total growth was covered in the second experiment with and without metabolic activation.

The isolated minor reduction of the relative total growth to 43.5% in the first culture of experiment I with metabolic activation was not considered a real toxic effect since no comparable reduction was observed in the parallel culture under identical conditions.

No substantial and reproducible dose dependent increase of the mutation frequency was observed with and without metabolic activation. The mutation frequency did not reproducibly reach or exceed the threshold of 126 above the mutation frequency of the corresponding solvent control in any of the experimental parts. An isolated increase exceeding the threshold was noted in the first culture of experiment I without metabolic activation at 19.5 µg/mL. However, this increase was judged as irrelevant fluctuation since it was not reproduced in the parallel culture under identical experimental conditions. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance. In experiment II the mutant frequency exceeded the range of the historical solvent control data at several test points without metabolic activation (both cultures) and at one test point with metabolic activation (culture I). However, the threshold described above was not reached at any test point of the second experiment and no dose dependent increase was indicated by statistical analysis.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in all experimental groups. In this study the range of the solvent controls was from 130 up to 164 mutant colonies per 106cells; the range of the groups treated with the test item was from 83 up to 275 mutant colonies per 106cells. The solvent controls remained within the range of the historical data. Methylmethanesulfonate (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and cyclophosphamide (3.0 µg/mL and 4.5 µg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls.

There was no concentration related positive response of induced mutant colonies over background.

 

In vivo data

The cytogenetic activity of the AAPBs was tested in a OF1 (I. O. P. S. Caw) mouse bone marrow micronucleus assay, performed as described by W. Schmid - The Micronucleus test, Mutation Research, 31, 9-15 (1975). 5 male and 5 female animals were treated i. p. with Coco AAPB (30 % a. i). The test method by W. Schmidt is almost equivalent to the procedure described by OECD guideline 474. In a preliminary study the test animals were administered twice (in a 24 hours interval) each 100, 200, 500, 1000 and 2000 mg/kg bw/day by intraperitoneal injection. Clinical signs and mortality were observed up to 30 hours after the first administration. Clinical signs like piloerection and ptosis were seen at doses of ≥ 100 mg/kg bw/day. At doses ≥ 1000 mg/kg bw/day the mice died within 30 and 4 hours after the first administration. The tolerated doses were in the range of 100 to 500 mg/kg bw/day. Therefore, the dose of 200 mg/kg bw/day was selected as the high dose and 20 mg/kg bw/day (10 % of the high dose) as the low dose. As the test substance was applied twice with a 24 h interval (although only one timepoint was chosen for sacrifice), the result of the sacrifice 6h later may be regarded as a result of a 30h and a 6h treatment. The dose level of 200 mg/kg bw/day (corresponding to 60 mg active substance/kg bw/day) is considered to be sufficiently high based on the effects found in the preliminary study and due to the highly irritating properties of the compound.

The mean number of micronucleated erythrocytes/1000 polychromatic erythrocytes in males and female mice at 20 and 200 mg/kg bw/day were unaffected compared to the negative controls. The administration of 100 mg cyclophosphamide/kg bw serving as the positive control led to clearly elevated numbers of micronucleated erythrocytes.

It can be concluded, that Coco AAPB (30 % a. i) induced no clastogenic effect in this in vivo cytogenicity study on mice at dose levels of 20 and 200 mg/kg bw/day.

 

Conclusion

Reliable, relevant and adequate data on the genetic toxicity of AAPBs is available from several in vitro gene mutation studies in bacteria (Ames Tests), one in vitro gene mutation study in mammalian cells (L5178Y/ TK Mouse Lymphoma assay) and one in vivo cytogenicity study (in vivo Mammalian Erythrocyte Micronucleus Test). The full set of mutagenicity tests required by REACH Regulation Annexes VII and VIII is covered with the studies, whereas the information requirement on an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study is fulfilled in accordance to Annex VIII, column 2 by adequate data from an in vivo cytogenicity study. The studies were performed on C8-18 and C18 unsatd. AAPB or C8-18 AAPB.

There was no evidence of mutagenic or clastogenic intrinsic properties in any of the performed studies.

The genotoxic potential of the whole group of AAPBs is assumed to be similar. As fatty acids independently from their chain length and degree on unsaturation are generally considered to be not genotoxic, a variability in the fatty acid moiety is not expected to have any influence on the genotoxic activity of the AAPBs. Thus, the use of studies performed on individual members of this group of substances as read-across for the whole group is justified without restrictions.

In conclusion there is no evidence for genotoxic properties for C12 AAPB.

 

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 read-across

For details on substance identity and detailed toxicological profiles, please refer also to the general justification for read-across given at the beginning of the CSR and attached as pdf document to IUCLID section 13.

This read-across approach is justified based on structural similarities. All AAPBs contain the same functional groups. Thus a common mode of action can be assumed.

The only deviation within this group of substances is a minor variety in their fatty acid moiety (chain length and degree of unsaturation), which may have an influence on the outcome of skin and eye irritation studies, but is not expected to have any influence on genotoxic properties.

 

a. Structural similarity and functional groups

Alkylamidopropyl betaines (AAPBs) are – with the exception of C12 AAPB - UVCB substances (Substances of Unknown or Variable composition, Complex reaction products or Biological materials), which are defined as reaction products of natural fatty acids or oils with dimethylaminopropylamine and further reaction with sodium monochloroacetate. AAPBs are amphoteric surfactants, which are characterized by both acidic and alkaline properties.

 

Their general structure is:

 

R-C(O)-NH-(CH₂)₃-(N(CH₃)₂)⁺-CH₂-C(O)O^-

R = fatty acid moiety

 

The fatty acids have a mixed, slightly varying composition with an even numbered chain length from C8 to C18. Unsaturated C18 may be included. Consequently, the AAPBs differ by their carbon chain length distribution and the degree of unsaturation in the fatty acid moiety. However, Lauramidopropyl betaine (C12 fatty acid derivate) is the major ingredient of all AAPBs covered by this justification as listed in table 1 “Substance identities” of the general justification for read-across.

 

The substances under evaluation share structural similarities with common functional groups (quaternary amines, amide bonds and carboxymethyl groups), and fatty acid chains with differences in chain length and degree of saturation. As fatty acids independently from their chain length and degree on unsaturation are generally considered to be not genotoxic, a variability in the fatty acid moiety is not expected to have any influence on the genotoxic activity of the AAPBs.

 

b. Differences

Differences in acute toxicity of the AAPBs could potentially arise from the following facts:

-Different amounts of different carbon chain lengths (carbon chain length distribution):

Higher amounts of higher chain lengths and corresponding lower amounts of lower chain length could result in a rising average lipophilicity. As fatty acids independently from their chain length are generally considered to be not genotoxic, a variability in the fatty acid moiety is not expected to have any influence on the genotoxic activity of the AAPBs

- Different amounts of unsaturated fatty ester moieties:

Effects may be expected for e.g. physical state and for some toxicological endpoints, mainly local effects (e.g. irritation). However, as fatty acids independently from their degree on unsaturation are generally considered to be not genotoxic, a variability in the fatty acid moiety is not expected to have any influence on the genotoxic activity of the AAPBs.

 

Comparison of genotoxicity data

 

Endpoints	Source substances		Target substance
	C8-18 AAPB	C8-18 and C18 unsatd. AAPB	C12 AAPB
Genotoxicity in vitro Gene mutation in bacteria	No data	key_Genetic toxicity in vitro: 147170-44-3_8.4.1_Zschimmer_1996_EEC 92_69   key study   EU Method B.13/14, gene mutation: bacterial reverse mutation assay (e.g. Ames test), S. typhimurium TA 1535, TA 100, TA 1537, TA 1538 and TA 98 Metabolic activation: with and without cytotoxicity: first evidence of toxicity at 10000 µg/plate with and without S9   Genotoxicity: negative   Reliability: 2 (reliable with restrictions), no GLP	No data, read-across
		Sup_Genetic toxicity in vitro: 61789-40-0_8.4.1_Hüls_1995_OECD 471   Supporting study   OECD TG 471, gene mutation: bacterial reverse mutation assay (e.g. Ames test) Metabolic activation: with and without cytotoxicity: >= 1000 µg/plate (TA 1535, 1537), 5000 µg/plate (TA98)   Genotoxicity: negative   Reliability: 1 (reliable without restriction), yes
		Sup_Genetic toxicity in vitro: 61789-40-0_8.4.1_Henkel_1988_OECD 471   Supporting study   OECD TG 471, gene mutation: bacterial reverse mutation assay (e.g. Ames test) Metabolic activation: with and without cytotoxicity: >= 256 µg/plate   Genotoxicity negative   Reliability: 1 (reliable without restriction), GLP
		Sup_Genetic toxicity in vitro: 61789-40-0_8.4.1_REWO_1987_OECD 471_neuscan   Supporting study   OECD TG 471, gene mutation: bacterial reverse mutation assay (e.g. Ames test) S. typhimurium TA 100, TA 1535, TA 98, TA 1538, TA 1537 Metabolic activation: with and without cytotoxicity: no (3.12 µl/plate no colonies on the plates detected)   Genotoxicity negative   Reliability: 1 (reliable without restriction), GLP
		Sup_Genetic toxicity in vitro: 61789-40-0_8.4.1_THG_1982   Supporting study   similar to OECD TG 471, gene mutation: bacterial reverse mutation assay (e.g. Ames test) S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 Metabolic activation: with and without cytotoxicity: not determined   Genotoxicity: negative   Reliability: 2 (reliable with restrictions), no GLP
Gene mutation in mammalian cells	Key_Genetic toxicity in vitro: 97862-59-4_8.4.3_MoLy_Evonik_2010_OECD-476 key study OECD TG 476,gene mutation: mammalian cell gene mutation assay,  mouse lymphoma L5178Y cells Metabolic activation: with and without cytotoxicity: yes   Genotoxicity: negative   Reliability: 1 (reliable without restriction), GLP	No data	No data, read-across
Genotoxicity in vivo	No data	Key_Genetic toxicity in vivo: 61789-40-0_8.4.4_Goldschmidt_France_1987_micronucleus test in vivo Study result type: experimental result similar to OECD Guideline TG 474,chromosome aberration: micronucleus assay, mouse, intraperitoneal,100 mg/kg bw   toxicity: yes (in preliminary dose finding study lethal at doses >/= 1000 mg/kg bw); vehicle controls valid: yes; positive controls valid: yes Genotoxicity negative   Reliability: 2 (reliable with restrictions), GLP	No data, read-across

 

The source substance C8-18 and C18 unsatd. AAPB was not mutagenic in several reverse gene mutation assays in bacteria (Ames test).

In a mammalian cell gene mutation assay according to OECD Guideline 476 the source substance C8-18 AAPB induced no concentration related positive response of induced mutant colonies over background when tested up to cytotoxic concentrations.

The source substance C8-18 and C18 unsatd. AAPB was not clastogenic in a mouse bone marrow micronucleus assay.

 

Quality of the experimental data of the analogues:

The available data are adequate and sufficiently reliable to justify the read-across approach.

The key studies wereconducted according toEU Method B.13/14, OECD Guideline 476 or similar to OECD Guideline 474 and werereliable orreliable with restrictions (RL1-2).

Several supporting studies (RL1-2) are available, which wereconducted according to (or comparable to) OECD Guideline 471.

The test materials used in the respective studies represent the source substance as described in the hypothesis in terms of substance identity and minor constituents.

Overall, the study results are adequate for the purpose of classification and labelling and risk assessment.

 

Conclusion

Based on structural similarities of the target and source substancesas presented above and in more detail in the general justification for read across, it can be concluded that the available data from the source substances C8-18 AAPB as well asC8-18 and C18 unsatd. AAPB are also valid for the target substance C12 AAPB.

As fatty acids independently from their chain length and degree on unsaturation are generally considered to be not genotoxic, a variability in the fatty acid moiety is not expected to have any influence on the genotoxic activity of the AAPBs. Thus, the use of studies performed on individual members of this group of substances as read-across for the whole group is justified without restrictions.

There was no evidence of mutagenic or clastogenic intrinsic properties in any of the performed studies.

Justification for classification or non-classification

AAPBs considered to have no genotoxic properties as shown in several gene mutation studies in bacteria (Ames Tests), one in vitro gene mutation study in mammalian cells (L5178Y/ TK Mouse Lymphoma assay) and one in vivo cytogenicity study (in vivo Mammalian Erythrocyte Micronucleus Test).

Therefore, AAPBs do not comply with the classification requirements regarding germ cell mutagenicity outlined in regulation (EC) 1272/2008.