Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dibutylstannane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 12 May 2010 and 07 June 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted to GLP. Study refers to the tin compound of the reaction mass

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiment 1: 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.

Experiment 2: Salmonella strains: 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
E.coli strain WP2uvrA: 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in house. Acetone was therefore selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without S9: N-ethyl-N-nitro-N-nitrosoguanidine, 9-Aminoacridine & 4-Nitroquinoline-1-oxide; With S9: 2-Aminoanthracene & Benzo(a)pyrene.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: In experiment 2, measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or test material formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar.
- Exposure duration: All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: nda

DETERMINATION OF CYTOTOXICITY
- Method: In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The test material was, therefore, tested up to the toxic limit.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The appropriate characteristics for each tester strain have been confirmed, eg rfa cell wall mutation and pKM101 plasmid R-factor etc.
- All tester strain cultures should be in the range of 1 to 9.9 x 10^9 bacteria per ml.
- Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
A precipitate (greasy in appearance) was noted in the preliminary toxicity test at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
The test material was found to be initially toxic at and above 50 and 500 µg/plate to the strains of bacteria used TA100 and WP2uvrA-, respectively. The test material formulation and S9 mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, see appendix 2

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Preliminary Toxicity Test

The test material was initially toxic at and above 50 and 500 µg/plate to the strains of bacteria used TA100 and WP2uvrA^-, respectively. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	165	151	144	154	157	129	27S	0S	0T	0TP	0TP
+	TA100	169	150	159	148	155	133	12S	0S	0T	0TP	0TP
-	WP2uvrA^-	23	19	18	22	20	21	16	16	0T	0TP	0TP
+	WP2uvrA^-	33	24	34	21	24	14	11	11	0S	0TP	0TP

S = Sparse bacterial background lawn
T = Toxic, no bacterial background lawn
P = Precipitate

Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

A history profile of vehicle and positive control values for 2008 and 2009 is presented in Appendix 2.

In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, in both the presence and absence of S9-mix, at and above 150 and 500 µg/plate for the Salmonella strains and Escherichia coli strain WP2uvrA^-, respectively. A similar toxic response was noted in the second experiment (pre-incubation method) with weakened bacterial background lawns noted to all of the tester strains, initially from 50 µg/plate (TA100 and TA1535), 150 µg/plate (TA98 and TA1537) and 500 µg/plate for Escherichia coli strain WP2uvrA^-. The test material was, therefore, tested up to the toxic limit. A precipitate (greasy in appearance) was noted in the preliminary toxicity test at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA^- were treated with the test material using both the Ames plate incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 0.5 to 500 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The test material dose range was amended slightly (ranging between 0.15 and 500 µg/plate) to allow for results from Experiment 1 and the change in test methodology.

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, in both the presence and absence of S9-mix, at and above 150 and 500 µg/plate for the Salmonella strains and Escherichia coli strain WP2uvrA^-, respectively. A similar toxic response was noted in the second experiment (pre-incubation method) with weakened bacterial background lawns noted to all of the tester strains, initially from 50 µg/plate (TA100 and TA1535), 150 µg/plate (TA98 and TA1537) and 500 µg/plate for Escherichia coli strain WP2uvrA^-. The test material was, therefore, tested up to the toxic limit. A precipitate (greasy in appearance) was noted in the preliminary toxicity test at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Data based on the report of the experimental results. Endpoint will be updated as soon as the final report is available

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

n.a.

Species / strain / cell type:

other: cultured human lymphocyte cells

Details on mammalian cell type (if applicable):

cultured human lymphocyte cells, sex: female

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

10.0 - 0.0363 µg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other:

Remarks:

CPA, 6.25 // NQO, 2.50 // concentration in µg/ml

Key result

Species / strain:

other: cultured human lymphocyte cell

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
positive

Wacker Catalyst 41 is cytotoxic and induced structural chromosome aberrations in cultured human lymphocyte cells when tested for 3+17 hours in the absence and presence of S-9.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Draft report in due to time frame for registration, update of this endpoint as soon as final version available

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

TA98: his D3052, rfa-, uvrB-, r-factor
TA100: his G46, rfa-, uvrB-, r-factor
TA1535: his G46, rfa-, uvrB-
TA1537: his C3076, rfa-, uvrB-,
TA102: his G428 (pAQ1), rfa-, r-factor

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

experiment I: 0.316, 1.00, 10.0, 31.6, 100, 316, 1000 µg/plate
experiment II: 0.158, 0.50, 5.0, 15.8, 50, 158, 500 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: without activation: for TA 100, TA 1535: sodium azide // for TA98, TA 1537: 4-nitro-o-phenylene-diamine // for TA 102: methylmethansulfonate // for all strains with activation: 2-aminoantharacene

Remarks:

solvent: EtOH

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

see attachment

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can stated that during the described mutagenicity test and unter the experimental conditions reportet, Wacker Katalysator 41 didi not cause mutations by base pair changes or frameshifts in the genome ot the tester strains used.
Therefore Wacker Katalysator 41 is considered to be non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

IN VITRO GENE MUTATION STUDY IN BACTERIA

 

Bowles & Thompson (2010), a GLP compliant study performed to the OECD guideline 471 was included as the key study for this data requirement. Accordingly the study was assigned a reliability score of 1 and considered reliable and adequate for assessment.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA^-were treated with the test material using both the Ames plate incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). In the plate incorporation test, a visible reduction was noted in the growth of the bacterial background lawns of all of the tester strains, in both the presence and absence of S9-mix, at and above 150 and 500 µg/plate for the Salmonella strains and Escherichia coli strain WP2uvrA^-, respectively. A similar toxic response was noted in the second experiment (pre-incubation method) with weakened bacterial background lawns noted to all of the tester strains, initially from 50 µg/plate (TA100 and TA1535), 150 µg/plate (TA98 and TA1537) and 500 µg/plate for Escherichia coli strain WP2uvrA^-. The test material was, therefore, tested up to the toxic limit. A precipitate (greasy in appearance) was noted in the preliminary toxicity test at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. The test material was considered to be non-mutagenic under the conditions of this test.

IN VITRO CYTOGENICITY STUDY IN MAMMALIAN CELLS OR IN VITRO MICRONUCLEUS STUDY

 

Reimann R & Gramlich U (1990) was provided as the key study for this data requirement. The study was performed in compliance with GLP and the method was comparable to that of OECD 473. The study was accordingly assigned a reliability score of 2 and considered reliable and adequate for assessment. An evaluation of the clastogenic potential in the human lymphocyte test indicated clastogenic potential of the test material in the human lymphocyte test in vitro at clearly cytotoxic concentrations. From the four assays conducted without and with an extrinsic metabolizing system in two independent studies, one assay without and one with S9 mix gave statistically significant (P < 0.05) increases in the frequency of chromosomal aberrations at the highest concentrations evaluated, whereby in the remaining assays the results were borderline negative. In each assay of this investigation, the test material was tested up to cytotoxic concentrations as indicated by an obvious reduction of the mitotic index.

 

The CHO gene mutation study from the publication by Li AP et al (1982) was provided as a supporting study to this endpoint. The study was conducted to good scientific principles (GLP status was not reported), however the study did not include metabolic activation. The study was accordingly assigned a reliability score of 2. The LC50 value of DBTC for CHO cells, as determined by cloning efficiency, was approximately 0.35 µg/ml (1.12 µM). DBTC induced mutations at the HGPRT gene locus in CHO cells. The mutant frequency increased with dose up to 0.2 µg/ml (0.66 µM) for DBTC. A decrease in mutant frequency was observed at higher concentrations.

 

IN VITRO GENE MUTATION STUDY IN MAMMALIAN CELLS

 

Lang R & Schmitt R (1989) was provided as the key study for this data requirement. The study was well documented, performed in compliance with GLP and to a method comparable to OECD 476. The study was assigned a reliability score of 2 and considered reliable and adequate for use. The study was a HGPRT-test with V79 cells. The test material was found to have cytotoxic effects without metabolic activation by S9 mix at 0.00006 µl/ml and with metabolic activation a clear toxic effect could be observed at 0.0003 µl/ml in the first experiment and at 0.0005 µl/ml in the second assay of the second experiment. The test material did not show a mutagenic potential in the HGPRT/V79 mammalian cell gene mutation test neither in the absence nor in the presence of rat liver S9 mix in two independently performed experiments.

 

IN VIVO MUTAGENICITY

 

Dance C (1991) was provided as the key study for this data requirement. The study was performed in compliance with GLP and according to the guideline OECD 474 (and EU Method B.12). The study was therefore assigned a reliability score of 1 and considered reliable and adequate for assessment. The study investigated the clastogenic action on bone marrow erythrocytes in a micronucleus. The test material showed evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of mice treated orally with DBTC at 50 mg/kg and sacrificed 48 or 72 hours later. A biologically and statistically significant increase in the incidence of micronucleated polychromatic cells was observed in the bone marrow of mice treated with DBTC at 50 mg/kg and killed 48 and 72 hours later (0.01<p<0.05): this effect was seen more clearly in females than in males. No such effect was apparent for any group treated with DBTC and killed 24 hours later (p>0.05). Statistically significant increases over controls were also seen in positive control group animals given chlorambucil at 30 mg/kg (p<0.01).

 

A further in vivo mutagenicity study, (Lang R & Wedel JV (1990) was provided as supporting information. The GLP status of the study was not reported and no guidelines were listed. The study was performed to a good scientific standard with a good level of reporting of the methodology and the results. The study investigated the mutagenic potential of the test material in the mouse micronucleus test. The test material failed to show any evidence of mutagenic potential, when administered by gavage up to the toxic dose level of 200 mg/kg in the mouse micronucleus test. Triaziquone, the positive reference, gave the expected mutagenic response. After application of the high dose four males and one female died; after application of the mid dose, one male died. More than half of the animals of the two highest dose groups showed signs of toxicity (e.g. apathy, eyelid closure, ruffled fur).

 

OTHER:

In the second part to the Li AP et al (1982) study, a test was performed to determine the cytotoxicity of the test material to rat lymphocytes. The test is not a standard endpoint and was therefore provided for information purposes only. The study was performed to a good scientific standard with a good level of reporting. The LC50 for lymphocytes as determined by dye-exclusion was approximately 50 µg/ml (0.16 mM). At the same concentration of DBTC, the number of antibody-forming cells (AFC) was reduced to approximately 10 % of the control.

Overall summary:

The SPCFC (2004) review of organotins concluded that (organotins in general) did not exhibit any significant genotoxic potential in vivo, and that carcinogenicity seen with some organotin compounds was likely attributable to hormonal or immunotoxic actions. Dibutyltin salts are recommended for classification as mutagenic (R68) within the EU classification system. The available data for dibutyltin oxide and dibutyltin chloride as described above are inadequate to challenge that recommendation.

Further the study form Lloyd et al. shwoed the the mutagenic properties in the reaction mass from the dibutlytin are still existing.

Short description of key information:
The following studies were submitted as key studies to address the genetic toxicity of the substance:

IN VITRO GENE MUTATION STUDY IN BACTERIA

Bowles A & Thompson PW (2010) REVERSE MUTATION ASSAY “AMES TEST” USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI. Testing Laboratory: Harlan Laboratories Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK. Owner Company: Organo Tin REACH Consortium, c/o ReachCentrum, Avenue E. Van Nieuwenhuyse 6, B-1160, Brussels, BELGIUM. Report No.: 3109/0031

IN VITRO CYTOGENICITY STUDY IN MAMMALIAN CELLS OR IN VITRO MICRONUCLEUS STUDY

Reimann R & Gramlich U (1990). ZK 22.663: Evaluation of the clastogenic potential in the human lymphocyte test. Testing laboratory: Schering AG, Pharmaceutical Research, Bergkamen, Germany. Owner company: Schering AG, Pharmaceutical Research, Bergkamen, Germany. Report No.: IC 1/90. Report Date: 1990-09-17.

IN VITRO GENE MUTATION STUDY IN MAMMALIAN CELLS

Lang R & Schmitt R (1989). ZK 22.663: Evaluation of gene mutations in mammalian cells in culture: HGPRT-test with V79 cells. Testing laboratory: Schering AG, Pharmaceutical Research, Bergkamen, Germany. Owner company: Schering AG, Pharmaceutical Research, Bergkamen, Germany. Report No.: IC 16/89. Report date: 1989-03-30.

IN VIVO MUTAGENICITY

Dance C (1991). Dibutyl tin chloride: assessment of clastogenic action on bone marrow erythrocytes in the micronucleus test. Testing Laboratory: Life Sciences Research Limited, Eye, Suffolk, IP23 7PX, England. Owner company: Atochem North America Incorporated, 900 first Avenue, P.O. Box C, King of Prussia, Pennsylvania, 19406-0018, USA. Report No.: 91/0357. Report date: 1991-11-08

With the exception of Bowles & Thompson (2010) (assigned a reliability score of 1 and performed on dibutyltin di(acetate)), all the above listed studies were read-across from dibutyltin dichloride (a similar organotin compound) to the substance in question and assigned a reliability score of 2.

Endpoint Conclusion: Adverse effect observed (positive)

Justification for classification or non-classification

According to directive 67/548/EEC the substance is assigned the classification Mutagenicity category 3 and labelled with R68 – possible risk of irreversible effects. According to Regulation (EC) no 1272/2008 the test substance would be classified as Muta. 2 with the Hazard statement: H341: Suspected of causing genetic defects and should be accompanied with the signal word 'Warning'.