Hexanoic acid, 3,5,5-trimethyl-, tin(2+) salt (2:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro data from a bacterial reverse mutation assay (Ames Test) and a micronucleus assay in human lymphocytes are available for hexanoic acid, 3,5,5-trimethyl-, tin (2+)salt (2:1). Additional data are available for the dissociation products 3,5,5-trimethylhexanoic acid (HPRT test, Ames test and Chromosome aberration assay) and for Sn2+ as tin dichloride (Mouse Lymphoma assay and Ames test).

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-21 till 2012-05-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD guideline 487

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, adopted July 22, 2010, Guideline No. 487 “In vitro Mammalian Cell Micronucleus Test”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment IA: 6.3, 12.5, 25.0, 50.0, 100.0, 200.0, 250.0, 300.0, 350.0, 400.0, 450.0, 500.0, 750.0 µg/mL
Experiment II: 5.0, 10.0, 50.0, 200.0, 250.0, 300.0, 320.0, 340.0, 360.0, 380.0, 400.0, 500.0 µg/mL

Without metabolic activation:
Experiment IA: 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0, 75.0, 100.0, 125.0, 150.0, 200.0, 400.0 µg/mL
Experiment IB: 0.5, 1.0, 5.0, 10.0, 60.0, 80.0, 100.0, 110.0, 120.0, 130.0, 140.0, 150.0, 300.0 µg/mL
Experiment II: 0.1, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, 200.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 487

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: demecolcin

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Three independent experiments were performed. In Experiment IA the exposure period was 4 hours with and without S9 mix. In Experiment IB the exposure period was 4 hours without S9 mix. In Experiment II the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. Evaluation of two cultures per dose group.

METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 20 hours (- S9 mix)
- Expression phase after 4 hours treatment: 16 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 40 hours


CYTOKINESIS BLOCK (cytogenetic assays): Cytochalasin B 20 hours
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF BINUCLEATED CELLS EVALUATED: 1000 per culture


DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI)

Evaluation criteria:

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data (see ANNEX II) and/or
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of
micronucleated cells is observed.
Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered
together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological
relevance is discussed and/or a confirmatory experiment is performed.

Statistics:

Statistical significance can be confirmed by means of the Chi square test.

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment IA the exposure period was 4 hours with and without S9 mix. In Experiment IB the exposure period was 4 hours without S9 mix. In Experiment II the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis.
The highest treatment concentration in pre-test, 4320.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
Precipitation of the test item in the culture medium was observed in the absence of S9 mix in Experiment IA at 1.6 µg/mL and above and in Experiment IB at 60.0 µg/mL and above at the end of treatment. In the presence of S9 mix precipitation occurred at 12.5 µg/mL and above in Experiment IA. In Experiment II precipitation was observed at 50.0 µg/mL and above in the absence and at 10.0 µg/mL and above in the presence of S9 mix at the end of treatment. No relevant influence on osmolarity or pH value was observed.
In Experiment IA in the absence and presence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. Cytostasis of 34.6 and 44.1 % was observed at the highest evaluated concentrations. Cytotoxicity indicated by a reduced CBPI and expressed as cytostasis (%) was observed in Experiment IB in the absence of S9 mix (52.3 % and 52.1 %) and in Experiment II in the presence of S9 mix (54.2%) at the highest evaluated concentrations. In Experiment II in the absence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. However, cytostasis was approx. 45.8 % at the highest evaluable concentration. For not evaluable concentrations, showing clear cytotoxic effects the total number of cells was markedly reduced as noticed during precheck of the slides.

In Experiment IA, in the absence of S9 mix, one single statistically significant increase in the number of micronucleated cells was observed after treatment with 100.0 µg/mL (2.15 % micronucleated cells), clearly exceeding the range of the historical solvent control data (0.15 -1.40 % micronucleated cells) . In Experiment IB in the absence of S9 mix this finding could not be confirmed. In Experiment II after treatment with 50.0 µg/mL without S9 mix one statistically significant increase in the number of micronucleated cells (1.20 %) was observed combined with a dose-dependent increase. The value is in the range of the historical solvent control data (0.05 - 1.45 % micronucleated cells) and therefore the statistical significance has to be regarded as being biologically irrelevant. No relevant increase in micronucleated cells was observed in the presence of S9 mix.
In both experiments, either Demecolcin (75 ng/mL), MMC (2.0 µg/mL) or CPA (10.0 or 12.5 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in human lymphocytes in vitro when tested up to cytotoxic and/or precipitating concentrations.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Table2: Summary of results of the in vitro micronucleus test in human lymphocytes

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells
		in µg/mL	CBPI		in %**
Exposure period 4 hrs without S9 mix
IA	40 hrs	Negative control	2.02		0.70
		Solvent control^1	2.00		1.20
		Positive control^2	1.67	34.7	12.20^S
		0.8	2.02	n.c.	1.25
		1.6^P	2.11	n.c.	1.25
		75.0^P	1.81	19.2	1.05
		100.0^P	1.65	34.6	2.15^S***
IB	40 hrs	Negative control	1.76		0.45
		Solvent control^1	1.94		0.65
		Positive control^2	1.60	20.9	6.40^S
		10.0	1.79	15.8	0.80
		60.0^P	1.70	25.2	0.65
		80.0^P	1.69	27.0	0.60
		100.0^P	1.45	52.3	0.70
		110.0^P	1.45	52.1	0.60
Exposure period 20 hrs without S9 mix
II	40 hrs	Negative control	1.97		0.10
		Solvent control^1	1.91		0.15
		Positive control^3	1.55	42.7	3.00^S
		12.5	1.92	n.c.	0.30
		25.0	1.75	17.2	0.45
		50.0^P	1.49	45.8	1.20^S

*       For the positive control groups, the relative values are related to the negative controls;
for the test item treatment groups the values are related to the solvent controls

**     The number of micronucleated cells was determined in a sample of 2000 binucleated cells

***   The number of micronucleated cells was determined in a sample of 4000 binucleated cells

n.c.  Not calculated as the CBPI is eqal or higher than the solvent control value

^P      Precipitation occurred at the end of treatment

^S      The number of micronucleated cells is statistically significantly higher than corresponding control values

¹      DMSO             1.0 % (v/v)

²      MMC                2.0 µg/mL

³      Demecolcin  75.0 ng/mL

Table 2 (cont.): Summary of results of the in vitro micronucleus test in human lymphocytes

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells
		in µg/mL	CBPI		in %**
Exposure period 4 hrs with S9 mix
IA	40 hrs	Negative control	1.75		0.95
		Solvent control^1	2.02		0.80
		Positive control^2	1.77	n.c.	2.20^S
		6.3	1.91	10.6	0.70
		12.5^P	1.96	5.3	0.55
		200.0^P	1.85	16.0	0.95
		300.0^P	1.57	44.1	0.90
II	40 hrs	Negative control	1.90		0.60
		Solvent control^1	1.90		0.20
		Positive control^3	1.54	40.1	5.30^S
		5.0	1.99	n.c.	0.05
		10.0^P	1.96	n.c.	0.10
		200.0^P	1.76	16.4	0.20
		300.0^P	1.41	54.2	0.10

*       For the positive control groups, the relative values are related to the negative controls;
for the test item treatment groups the values are related to the solvent controls

**     The number of micronucleated cells was determined in a sample of 2000 binucleated cells

n.c.  Not calculated as the CBPI is eqal or higher than the solvent control value

^P      Precipitation occurred at the end of treatment

^S      The number of micronucleated cells is statistically significantly higher than corresponding control values

¹       DMSO    1.0 % (v/v)

²         CPA     10.0 µg/mL

³         CPA     12.5 µg/mL

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in human lymphocytes
in vitro.
Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic and/or precipitating
concentrations.

Executive summary:

The test item hexanoic acid, 3,5,5-trimethyl-, tin (2+)salt (2:1), dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. The following study design was performed:

	Without S9-Mix		With S9-Mix
	Exp.& IB	Exp. II	Exp.and II
Exposure period	4 hrs	20 hrs	4 hrs
Expression phase	16 hrs	-	16 hrs
Cytochalasin B exposure	20 hrs	20 hrs	20 hrs
Preparation interval	40 hrs	40 hrs	40 hrs
Total culture period	88 hrs	88 hrs	88 hrs

In each experimental group two parallel cultures were analysed. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

The highest applied concentration in the pre-test on toxicity (4320 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487. Precipitation of the test item in the culture medium was observed in the absence and presence of S9 mix at least at the highest evaluated concentration.

In Experiment IA in the absence and presence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment IB in the absence of S9 mix cytotoxicity was observed at the two highest evaluated concentrations and in Experiment II in the presence of S9 mix at the highest evaluated concentration. In Experiment II in the absence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. However, cytostasis was approx. 45.8 % at the highest evaluable concentration.

In Experiment IA, in the absence of S9 mix, one single statistically significant increase in the number of micronucleated cells was observed after treatment with 100.0 µg/mL. In Experiment IB in the absence of S9 mix this finding could not be confirmed. In Experiment II after treatment with 50.0 µg/mL one single statistically significant increase in the number of micronucleated cells increase and dose-dependency was observed. As the value lies in the range of the historical solvent control data, this finding is considered as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (α< 0.05) in cells with micronuclei.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In-vitro bacterial reverse mutagenicity test:

In a reverse gene mutation assay in bacteria according to OECD Guideline 471 and EU Method B.13/14, the strains TA 1535, TA 1537, TA 98 and TA of S. typhimurium and WP2 uvrA of E. coli were exposed to hexanoic acid, 3,5,5-trimethyl-, tin (2+)salt (2:1) in the presence and absence of mammalian metabolic activation. The test item was diluted in acetone at concentrations of 62, 185, 556, 1667 and 5000 µg / plate in the first experiment. An irreversible clouding was observed in the first experiment; therefore ethylene glycol dimethyl ether was used as solvent in the second experiment.  

In both experiments precipitations were visible on the agar plates from 1667 µg/plate upwards. Toxicity of the highest test concentration was observed in all Salmonella typhimurium strains in both experiments and a varying degree in lower concentrations depending on Salmonella strain and activation status. No cytotoxicity was observed in the tester strain E. coli WP2 uvrA.

The test item did not induce gene mutations in the Salmonella tester strains with and without mammalian metabolic activation up to and including the highest dose of 5000 µg/plate.

In E. coli WP2 uvrA a dose depending slight increase of revertants was observed, 2.7, 2.3 and 2.1, 3.3 at 5000 µg/plate with and without mammalian metabolic activation, respectively. However, for this tester strain the mean values of solvent controls with and without metabolic activation were clearly below of historical control mean values.

In conclusion, regarding the low mean values of controls compared to historical control data and the only slight increase of revertants, the results of this bacterial reverse mutation assay were considered ambiguous.

In addition data from the read-across substances tin dichloride dehydrate and 3,5,5-trimethylhexanoic acid are available.

In a reverse gene mutation assay in bacteria the strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 of S. typhimurium and E. coli WP2 were exposed to tin dichloride dihydrate in the presence and absence of mammalian metabolic activation at concentrations of 0.033, 0.10, 0.33, 1.0, 3.3 and 10 mg per plate.

The test item did not induce increased revertant counts in all tester strains with and without mammalian metabolic activation up to and including the highest dose of 10 000 µg/plate.

Similar findings were observed with 3,5,5-trimethylhexanoic acid in an Ames-test with the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and E.coli WP2uvrA in concentrations up to 10000 µg/plate, both with and without metabolic activation

In-vitro chromosome aberration

For 3,5,5 -trimethylhexanoic acid a IUCLID robust study summary from an in-vitro mammalian chromosome aberration test is available. CHO cells were exposed to the test substance at concentrations of 12.5, 40, 125, 400 and 1250 µg/ml for 19 h. One half of the cells was analysed directly afterwards (19 h harvest, initial and repeated experiment), the other incubated with medium for a total of 43 h after start of exposure (43 h harvest, only repeated experiment).

The test substance did not induce chromosomal aberrations in the initial experiment (only 19 h harvest). The results for the second experiment were inconsistent and conflicting. As, in spite of every effort, it was not possible to make the study report available for evaluation no final conclusion can be drawn from this data.

 

In-vitro micronucleus test

An in-vitro micronucleus test with hexanoic acid, 3,5,5-trimethyl-, tin (2+)salt (2:1) was conducted according to OECD guideline 487.

In three independent experiments the test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

In Experiment IA the exposure period was 4 hours with and without S9 mix. In Experiment IB the exposure period was 4 hours without S9 mix. In Experiment II the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In Experiment IA in the absence and presence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment IB in the absence of S9 mix cytotoxicity was observed at the two highest evaluated concentrations and in Experiment II in the presence of S9 mix at the highest evaluated concentration. In Experiment II in the absence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. However, cytostasis was approx. 45.8 % at the highest evaluable concentration.

Precipitation of the test item in the culture medium was observed in the absence and presence of S9 mix at least at the highest evaluated concentration.

In Experiment IA, in the absence of S9 mix, one single statistically significant increase in the number of micronucleated cells was observed after treatment with 100.0 µg/mL. In Experiment IB in the absence of S9 mix this finding could not be confirmed. In Experiment II after treatment with 50.0 µg/mL one single statistically significant increase in the number of micronucleated cells increase and dose-dependency was observed. As the value lies in the range of the historical solvent control data, this finding is considered as biologically irrelevant.

 

 

Mutagenic activity in mammalian cells

In a mammalian gene mutation assay, mouse lymphoma cells (L5178 Y; thymidine kinase locus) cultured in vitro were exposed to the read-across substance tin dichloride up to a precipitating concentration of 80 µg/mL. The induction of small and large mutant colony populations was analyzed in the present and absence of mammalian metabolic activation.

Some erratic increases in MF of 1.5 to 1.8 fold were observed and these changes bore no relation to toxicity and were not repeatable among the three non-activation assays and two S9 activation assays. The lowest average RTG values obtained for treatments with soluble concentrations ranged from 30 -35% without S9 to ca. 60% with S9. Precipitation was observed at 80 µg/mL in culture medium, and a slightly acidic pH shift in the medium was noted at 50 µg/mL.

The test chemical was dosed into medium from a solution in DMSO because it was not soluble in water, even at 100 µg/mL, which shifted the pH to 3.5. This observation suggested that the actual material tested was stannous chloride dihydrate, which forms an insoluble basic salt in excess water. Since tin dichloride itself is soluble in water, possibly different toxic (and mutagenic) properties would have been obtained had the test chemical been more soluble and accessible to the cells. Also, the reduced toxicity in the presence of S9 mix suggested that stannous chloride is capable of interacting (in as yet unknown ways) with metabolic activation systems.

In conclusion, in the L5178Y mouse lymphoma cell mutation assay, tin dichloride is considered to be non-mutagenic.

 

In a study conducted according to OECD guideline 476 the potential of 3,5,5 -trimethylhexanoic acid to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was investigated.

The first experiment with 4-hour exposure was performed at concentrations of 102.5; 205.0, 410.0, 820.0, 1093.3 and 1366.7 µg/mL and 205.0, 410.0, 820.0, 1230.0 and 1640.0 µg/mL without metabolic activation and with metabolic activation, respectively.

The second experiment was performed with a treatment time of 4 hours with S9 mix (750.0, 1000.0, 1250.0, 1500.0 and 1640.0 µg/mL) and 24 hours without metabolic activation (500.0, 750.0, 1000.0, 1250.0, 1500.0 and 1640.0 µg/mL). The highest concentration (1640.0 µg/mL) was equal to a molar concentration of about 10 mM and was adjusted to purity.

Under the experimental conditions of this study 3,5,5 -trimethylhexanoic acid did not induce gene mutations at the HPRT locus in V79 cells.

 

Conclusion

In conclusion the available data do not indicate a genotoxic intrinsic property of the hexanoic acid, 3,5,5-trimethyl-, tin (2+)salt (2:1) relevant to humans.

 

Read-across justification:

Hexanoic acid, 3,5,5-trimethyl-, tin (2+) salt (2:1) is a tin-salt of an organic acid andis known to dissociatein aqueous medium. This assumption is sustained by a similar toxicological profile for hexanoic acid, 3,5,5-trimethyl-, tin (2+) salt (2:1) and 3,5,5 - trimethylhexanonic acid.

Hexanoic acid, 3,5,5-trimethyl-, tin (2+) salt (2:1) as well as 3,5,5 - trimethylhexanonic acid are both classified for acute oral toxicity in GHS category IV.

If the tin 2+species from adissociated salt isintroduced to aerated water, it will primarily be present as the poorly soluble Sn(OH)2species, or will beoxidized to become insoluble Sn(IV)O2.

In conclusion the hydrolysis products tin 2 +and the anion of the 3,5,5 - trimethylhexanonic acid can be regarded as surrogate substances for hexanoic acid, 3,5,5-trimethyl-, tin (2+) salt (2:1). Intrinsic properties of the dissociated ions are assumed to be independent of the source. Therefore it is reasonable to discuss toxicological effects of the ions separately. The 3,5,5 - trimethylhexanonic acid and for the tin 2+ moiety, tin dichloride, as a tin salt with a nontoxic counterion are considered appropriate for assessment of hexanoic acid, 3,5,5-trimethyl-, tin (2+) salt (2:1) and read-across is justified without restrictions.

Justification for selection of genetic toxicity endpoint
Data from a GLP compliant guideline study with reliability 1

Justification for classification or non-classification

In conclusion, hexanoic acid, 3,5,5-trimethyl-, tin (2+)salt (2:1) does not need to be classified as “genotoxic” according to Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008. No labelling is required.