Bis(dodecylthio)dioctylstannane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene Mutation study in Bacteria

(Read-Across Studies performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))

Findings from the 3 studies that are available on the read across substance are considered together in a weight of evidence approach to conclude that the registered substance is unlikely to be mutagenic when tested under the Ames test either with or without metabolic activation.

Supporting information is available on the read across substance (a mixture of DOTE, CAS 15571-58-1 and MOTE, CAS 27107-89-7, and 70:30).

Mouse Lymphoma Assay

(Read-Across Study performed on structurally similar substance (DOTE)(CAS No 15571 -58 -1))

It is concluded that under the conditions used in the study, the test material DOTE is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine locus

Species / strain / cell type:

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

Species / strain / cell type:

Saccharomyces cerevisiae

Remarks:

Strain D4

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

5.000, 1.000, 0.100, 0.010, 0.001 µL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water or DMSO
- Methylnitrosoguanidine: Water
- 2-Nitrofluorene: DMO
- Quinacrine mustard: Water
- 2-Anthramine: DMSO
- 2-Acetylaminofluorene: DMSO
- 8-Aminoquinoline: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water/ DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: Methylnitrosoguanidine, Quinacrine mustard, 2-Anthramine, 2-Acetylaminofluorene, 8-Aminoquinoline

Details on test system and experimental conditions:

- Approximately 10^8 cells from an overnight culture of each indicator strain were added to separate test tubes containing 2.0 mL of molten agar supplemented with biotin and a trace of histidine. For non-activation tests, at least four dose levels of the test material were added to the contents of the appropriate tubes and poured over the surfaces of selective agar plates. In activation tests, a minimum of four different concentrations of the test material were added to the appropriate tubes with cells. Just prior to pouring, an aliquot of reaction mixture (0.5 mL containing the 9,000 x g liver homogenate) was added to each of the activation overlay tubes, which were then mixed, and the contents poured over the surface of a minimal agar plate and allowed to solidify.

- The plates were incubated for 48 hours at 37 °C, and scored for the number of colonies growing on each plate.

- Positive and solvent controls using both directly active positive chemicals and those that require metabolic activation were run with each assay.

- The numbers of colonies on each plate were counted and recorded on printed forms. These raw data were analysed in a computer program and reported on a printout.

Evaluation criteria:

Evaluation Criteria for Ames Assay:
Because the procedures used to evaluate the mutagenicity of the test chemical are semi-quantitative, the criteria used to determine positive effects are inherently subjective and are based primarily on a historical data base. Most data sets are evaluated using the following criteria:
- Strains TA1535, TA1537, and TA1538
If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic.
- Strains TA98, TA100, and D4
If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the highest increase equal to twice the solvent control value for TA100 and two to three times the solvent control value for strains TA98 and D4 is considered to be mutagenic. For these strains, the dose response increase should start at approximately the solvent control value.
- Pattern
Because TA1535 and TA100 were both derived from the same parental strain (G-46) and because TA1538 and TA98 were both derived from the same parental strain (D3052), there is a built-in redundancy in the microbial assay. In general the two strains of a set respond to the same mutagen and such a pattern is sought. It is also anticipated that if a given strain, e.g. TA1537, responds to a mutagen in non-activation tests it will generally do so in activation tests. (The converse of this relationship is not expected.) While similar response patterns are not required for all mutagens, they can be used to enhance the reliability of an evaluation decision.
- Reproducibility
If a chemical produces a response in a single test that cannot be reproduced in one or more additional runs, the initial positive test data loses significance.

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Saccharomyces cerevisiae

Remarks:

D

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TOXICITY TEST RESULTS
The test material was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The dose range employed for the evaluation of this compound was from 0.001 to 5 µL/ plate.

WITHOUT METABOLIC ACTIVATION
The results of the tests conducted on the test material in the absence of a metabolic system were all negative.

WITH METABOLIC ACTIVATION
The results of the tests conducted on the test material in the presence of the rat liver activation system were all negative.

CONCLUSIONS
The test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic under these test conditions.

Table 1: Summary of Experiment

± S9 Mix	Concentration (µL/plate)	Revertants/plate
		TA1535	TA1537	TA1538	TA98	TA100	D4
-	Solvent 0.001 0.010 0.100 1.000 5.000	31 50 25 38 31 30	6 4 6 9 9 7	20 11 21 21 16 17	42 21 31 38 52 33	201 211 194 204 204 319	42 51 32 50 40 40
+	Solvent 0.001 0.010 0.100 1.000 5.000	35 38 31 14 16 23	14 18 21 14 12 7	25 17 30 25 35 21	51 45 42 40 49 43	226 224 237 243 254 272	44 46 47 38 45 42
Positive Controls
-	Name	MNNG	QM	NF	NF	MNNG	MNNG
	Concentration (µg/plate)	10	10	100	100	10	10
	Revertants/plate	935	448	834	939	>1000	925
+	Name	ANTH	AMQ	AAF	AAF	ANTH	DMNA
	Concentration (µg/plate)	100	100	100	100	100	100(µM)
	Revertants/plate	402	208	451	791	>1000	68

MNNG = Methylnitrosoguanidine

QM = Quinacrine mustard

ANTH = 2-Anthramine

NF = 2-Nitrofluorene

AAF = 2-Acetylaminofluorene

AMQ = 8-Aminoquinoline

Conclusions:

Under the conditions of the study, the test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic with or without metabolic activation.

Executive summary:

The genetic toxicity of the test material was investigated in a bacterial reverse mutation assay, broadly similar to OECD 471.

The test material was tested at 5.000, 1.000, 0.100, 0.010, 0.001 µL/plate both with and without metabolic activation, in the form of S9-mix. The following bacterial strains were used: Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA1538, and Saccharomyces cerevisiae strain D4. The overlay method of application was used.

The plates were incubated for 48 hours at 37°C, and scored for the number of colonies growing on each plate. Positive and solvent controls using both directly active positive chemicals and those that require metabolic activation were run with each assay. The numbers of colonies on each plate were counted and recorded.

The test material was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The results of the tests conducted on the test material in the absence of a metabolic system were all negative. The results of the tests conducted on the test material in the presence of the rat liver activation system were all negative.

Under the conditions of the study, the test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic with or without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 1977 to June 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The 1978 study was repeated with TA100 at higher test material concentrations due to the slightly increased (and dose-dependent) number of mutants of Salmonella typhimurium TA 100 in the highest tested concentration in the absence of metabolic activation

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- 0.5, 1, 2, 4, 8, 16, 32, 64 µL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: Anthracen-2-amine

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

precipitate in agar at highest tested concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Nonactivation Test Results: The results of the tests conducted on the compound in the absence of a metabolic system were all negative.
- Activation Test Results: The results of the tests conducted on the compound in the presence of the rat liver activation system were all negative.

Table 1: Results

		Salmonella typhimurium TA100
Substance	Conc. (µg/plate)	with S9	without S9
DMSO (vehicle control)	0	114	112
Anthracen-2-amin	2	770; 810	114
Benzo(a)pyrene	5	787	107
Test material	0.5	116	132
	1	113	152
	2	122	190
	4	142	244
	8	130	306
	16	136	513
	32	155	687
	64	42**	46**

** Lawn growth slightly impaired. Precipitates in agar.

Conclusions:

Under the conditions of this study the test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic under these test conditions.

Executive summary:

The 1978 study was repeated with TA100 at higher test material concentrations due to the slightly increased (and dose-dependent) number of mutants of Salmonella typhimurium TA 100 in the highest tested concentration in the absence of metabolic activation.

The results of the tests conducted on the test material in the absence of a metabolic system were all negative. The results of the tests conducted on the test material in the presence of the rat liver activation system were all negative.

Under the conditions of this study the test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic under these test conditions.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 December 2009 to 02 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

Target gene:

TK gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The chromosome number of these cells is 40 (stable aneuploid karyotype, 2n = 40).
The cells were stored as frozen stock cultures in liquid nitrogen.
Each new stock culture is checked for mycoplasma contamination, which was absent.
Cell culturing : The L5178Y cells were grown in culture medium consisting of RPMI 1640 medium (with HEPES and Glutamax-I) supplemented with heat-inactivated horse serum (10 % v/v for growing in flasks, and 20 % for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin.
On the day of exposure, the growth rate (doubling time of 9-14 h) and viability (>90 %; by trypan blue exclusion) of the cells were checked.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate

Test concentrations with justification for top dose:

FIRST test: Without S9 (24hr of treatment): 0.006-10 µg/ml ; With S9 (4 hr of treatment) : 0.01-20 µg/ml
SECOND test: Without S9 (4hr of treatment): 0.86-20 µg/ml; Without S9 (24hr) : 0.02-0.40µg/ml; With S9 : 1.6-60 µg/ml
THIRD test: Without S9 (24hr of treatment): 0.007-0.5 µg/ml ; With S9 (4hr of treatment): 1.4-100 µg/ml
With S9 (Third test, 4 hr of treatment): 1.4-100 µg/ml (11 concentrations)

Vehicle / solvent:

Ethanol.
The test material was diluted in ethanol to concentrations ranging from 0.04 to 10 mg/ml DOTE. From these stock solutions serial dilutions in ethanol were made and from each of these 0.1 ml were added to a final volume of 10 ml culture medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(ethanol)

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 4 hours or 24 hours (37°C, 5% CO2)
- Exposure duration: 20-24, and 44-48 hours (37°C, 5% CO2)
- Expression time (cells in growth medium): doubling time of 9-14 hours
- Selection time (if incubation with a selection agent): no data

NUMBER OF REPLICATIONS: single culture for each dose, 2 for negative control group, 1 for positive control group

NUMBER OF CELLS EVALUATED: 2000/well

DETERMINATION OF CYTOTOXICITY
- Method: by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG)

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no

Evaluation criteria:

The following criteria were used to validate the data obtained in the gene mutation assay (Cole et al., 1990; Aaron et al. 1994; Clive et al., 1995):
a) the average cloning efficiency of the negative controls should not be less than 60 % or more than 140 %.
b) the average suspension growth of the negative controls should be between 8 and 32.
c) the average mutant frequency of the negative controls should fall within the range of 40-300 TFT-resistant mutants per 1,000,000 clonable cells.
d) the mutant frequency of the positive controls should be higher than 400 TFTresistant mutants per 1,000,000 clonable cells, and should be at least twice that of the corresponding negative control.
e) unless the test substance shows no cytotoxicity at the highest possible concentration (determined by its solubility, pH and osmolar effects), the highest concentration should result in a clear cytotoxic response. The RTG value of one of the data points should be between 10 and 20 %, or one data point between 1 and 10% and another between 20 and 30 %.

The test material was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.
The test material was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test material concentrations.

Statistics:

no statistical analysis was performed.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

But increase at 72 µg/ml but this increase is not indicative for mutagenicity.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Results without S9 (see tables 1 and 2) :
In the absence of S9-mix three assays were performed in which mutagenicity was evaluated; two with 24 hours exposure and one with 4 hours exposure. In the first assay with 24 hours exposure the RTG at the highest concentration was 22 % (not between 10 and 20 %) and only six concentrations could be evaluated due to severe cytotoxicity. In the second assay with 24 hours exposure (results obtained in third test), sufficient cytotoxicity was observed at the highest concentration (RTG of 13 %) and nine concentrations could be evaluated resulting in clear to hardly any cytotoxic response. In the assay with 4 hours exposure (second test) at and above 5 μg/mL no clear dose response in cytotoxicity was observed, however, sufficient toxicity (RTG between 10 and 20 %) was observed for a valid test.
In none of the three assays performed in the absence of S9-mix any increase in mutant frequency (MF) by more than 88 or 126 mutants per 1,000,000 clonable cells, i. e. no equivocal or positive response, compared to the negative control was observed at any dose level.

Results with S9 (see table 3) :
In the presence of S9-mix two assays were performed in which mutagenicity was evaluated. In the first assay not sufficient cytotoxicity was observed at the highest concentration of 20 μg/mL (RTG was 38 %; not between 10 and 20 %). In the second assay (results obtained in third test), although relatively small intervals between the concentrations were used, none of the concentrations resulted in an RTG between 10 and 20 %. Above 44 μg/mL no dose response in cytotoxicity was observed. The RTG at 85 μg/ml was 30% whereas at 72 μg/ml the RTG was 5%. In addition, the relative suspension growth (RSG) at 61 and 85 μg/mL were 24 and 21%, respectively. This means that already in this phase almost 80 % cytotoxicity was observed, however, since the initial cell yield and cloning efficiencies at these concentrations were above 100 % (which is very unusual) this resulted not in RTG values below 20 %. In the presence of S9-mix at 72 μg/mL the mutant frequency was significantly increased by 238 mutants per 1,000,000 clonable cells compared to the negative control. Since relatively small intervals (0.85) were used and the increase was observed at a single concentration causing more than 90% cytotoxicity, it is concluded that this increase is not indicative for mutagenicity.

Colony sizing :
Colony sizing was performed in the third test at 2 concentrations in the presence of S9-mix, since a positive response in mutant frequency was observed. Both at 72 and 85 μg/mL relatively more small then large colonies were formed; 68 and 65 % compared to 32 and 35 %, respectively.
Since the increase in mutant frequency at 72 μg/mL was considered to be not relevant, no conclusions were drawn from these findings.

Positive and negative controls :
Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively; Ethanol served as negative control. The negative controls were within historical background ranges and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls.

RANGE-FINDING/SCREENING STUDIES:
Prior to the main study two dose range finding studies were performed; single cultures were exposed for 24 hours in the absence of S9-mix to 5 concentrations of DOTE ranging from 312 to 5000 μg/mL and 9.4 to 150 μg/mL, in the first and second study, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative controls were within historical background ranges and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls.

Table 1 : Summary of the results after 24 hours exposure in the absence of S9-mix.

Dose (µg/mL)	First assay (first test)		Dose (µg/mL)	First assay (first test)
	MF	RTG		MF	RTG
			0.36	115	13
			0.31	64	29
			0.26	63	36
0.2	89	22	0.22	93	60
0.1	72	81	0.16	115	79
0.05	76	95	0.11	59	100
0.03	57	84	0.079	93	99
0.01	74	96	0.055	80	108
0.006	58	81	0.027	72	110
0	93*	100*	0	74*	100*

*Mean of duplicate cultures.

MF = mutant frequency ; RTG = Relative total growth.

Table 2 : Summary of the results after 4 hours exposure in the absence of S9-mix.

Dose (µg/mL)	Second test
	MF	RTG
20	105	20
17	88	9
14	154	11
10	121	20
7.3	72	21
5.1	81	21
3.6	124	30
1.8	105	42
0.86	76	64
0	66*	100*

*Mean of duplicate cultures.

MF = mutant frequency ; RTG = Relative total growth.

Table 3 : Summary of the results after 4 hours exposure in the presence of S9-mix.

Dose (µg/mL)	First assay (first test)		Dose (µg/mL)	Second assay (third test)
	MF	RTG		MF	RTG
			85	143	30
20	117	38	72	318	5
14	105	49	61	89	39
9.8	83	60	44	111	35
6.9	80	80	32	140	42
3.4	73	88	22	71	57
1.6	75	93	11	103	85
0.8	69	101	5.4	83	89
0.4	87	84	2.7	95	83
0.2	78	93	1.4	100	71
0	86*	100*	0	80*	100*

*Mean of duplicate cultures.

MF = mutant frequency ; RTG = Relative total growth.

Conclusions:

It is concluded that under the conditions used in this study, the test material DOTE is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.
The test material DOTE was tested and evaluated for mutagenicity in both the absence and presence of metabolic activation (S9-mix). In the absence of S9-mix beside treatment for 4 hours, an extended treatment for 24 hours was used. In the three assays performed sufficient cytotoxicity was observed to validate the concentrations used and in none of the assays any indication for a mutagenic potential was observed.

Executive summary:

The test material DOTE was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). Three tests were conducted; in the first and third test single cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively; in the second test cultures were treated for 24 hours and 4 hours in the absence of S9-mix and 4 hours in the presence of S9-mix. The test material was dissolved in ethanol prior to testing.

The highest concentrations of DOTE evaluated for mutagenicity in the absence of S9-mix, were 20 μg/mL and 0.36 μg/mL at 4 and 24 hours exposure, respectively. In the presence of S9-mix and 4 hours exposure, the highest concentration evaluated was 85 μg/mL. The maximum concentrations were limited by cytotoxicity.

DOTE was cytotoxic in both the absence and presence of S9-mix. In the absence of S9-mix and 24 hours exposure cytotoxicity, resulting in a reduction in initial cell yield and/or suspension growth, was observed at and above 0.1 μg/mL. The relative total growth (RTG) at the highest concentration evaluated in the first and third test were 22 and 13 %, respectively. In the absence of S9-mix and 4 hours exposure cytotoxicity was observed at all concentrations included (as from 0.86 μg/mL). The relative total growth (RTG) at the highest concentration evaluated in the second test was 20 %.

In the presence of S9-mix cytotoxicity was observed at and above 1.4 μg/mL. The relative total growth (RTG) at the highest concentration evaluated in the first and third test were 38 and 30 %, respectively. In the absence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. In the presence of S9-mix at 72 μg/mL the mutant frequency was significantly increased by 238 mutants per 1,000,000 clonable cells compared to the negative control. Since relatively small intervals (0.85) were used and the increase was observed at a single concentration causing more than 90 % cytotoxicity compared to six concentrations causing 50-70 % cytotoxicity which showed no increase in mutant frequency, it is concluded that this increase is not indicative for mutagenicity. It is very likely a secondary effect to cytotoxicity and thus not indicative for mutagenicity.

Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and presence of the S9-mix, respectively; ethanol (vehicle) served as negative control. The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls.

It is concluded that under the conditions used in this study, the test material DOTE is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Three hundred chemicals were tested for mutagenicity, under code, in Salmonella typhimurium, using a preincubation protocol. All tests were performed in the absence of exogenous metabolic activation, and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

His

Species / strain / cell type:

other: Salmonella typhimunum strains TA97, TA98, TA100, TA1535, and TA1537

Details on mammalian cell type (if applicable):

- Source: Dr. Bruce Ames (University of California, Berkeley)
- Cultures were grown overnight with shaking at 37 °C in Oxoid No. 2 broth, and their phenotypes were analysed prior to their use for mutagenicity assays.

Metabolic activation:

with and without

Metabolic activation system:

S-9 fractions

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine for TA98 in the absence of metabolic activation.With metabolic activation for all strains: 2-aminoanthracene

Remarks:

sodium azide (TA1535 and TA 100), 9-aminoacridine (TA97 and TA 1537), and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.

Details on test system and experimental conditions:

TEST PROCEDURE
- The pre-incubation assay was performed, with some differences, as described below. The test chemical (0.05 mL), Salmonella culture (0.10 mL), and S-9 mix or buffer (0.50 mL) were incubated at 37 °C, without shaking, for 20 min. Chemicals known or suspected to be volatile were incubated in capped tubes. The top agar was added and the contents of the tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium.
- The histidine-independent (his') colonies arising on these plates were counted following two days incubation at 37 °C. Plates were machine counted unless precipitate was present which interfered with the count, or the colour of the test chemical on the plate reduced the contrast between the colonies and the background agar. At the discretion of the investigators, plates with low numbers of colonies were counted by hand.
- Variations in the protocol among the tested chemicals reflect the evolution of the protocol originally described by Haworth et al. Four protocol variations are evident from the data.
1) Testing in strains TA97, TA98, TA100, and TA1535, with some additional testing in strain TA1537; 10 % S-9 was used.
2)The first test of a chemical was without activation and with 10 % S-9 in the S-9 mix. If a positive result was obtained the test was repeated. If the tests were negative they were repeated without S-9 and with 30 % S-9.
3) The order of use of 10 % and 30 % S-9 was reversed.
4) Initial testing was in strains TA98 and TA100 without activation and with 30 % rat and hamster S-9s. If a positive result was obtained in one of these two strains it was repeated and the other strains were not used. If the tests were negative, the other strains were used with 30 % and 10 % S-9. A chemical was not designated non-mutagenic unless it had been tested in strains TA98, TA100, TA1535, and TA97 and/or TA1537, without activation and with 10 % and 30 % rat and hamster S9. Occasionally, 5 % S-9 was also used in all protocol variations.
- All chemicals were tested initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100 or the system developed by Waleh et al. [1982]. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.
- Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in the preliminary toxicity test. Subsequent trials occasionally used narrower dose increments and may not have included doses in the toxic range. Chemicals that were not toxic were tested, with few exceptions, to a maximum dose of 10 mg/plate. Chemicals that were poorly soluble were tested up to doses defined by their solubility’s. At least five doses of each chemical were tested in triplicate. Experiments were repeated at least one week following the initial trial. A maximum of 0.05 mL solvent was added to each plate.
- Concurrent solvent and positive controls were run with each trial. The positive controls in the absence of metabolic activation were sodium azide (TA1535 and TA 100), 9-aminoacridine (TA97 and TA 1537), and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene. Occasionally, a laboratory used a single solvent and/or positive control for more than one chemical tested on the same day. Each laboratory made its own determination regarding positive control dose levels.

Evaluation criteria:

DATA EVALUATION
- Evaluations were made at both the individual trial and overall chemical levels. Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+W,” if only a single dose was elevated over the control, or if the increase seen was not dose related. The distinctions between a questionable mutagenic response and a non-mutagenic or weak mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach twofold over background for a chemical to be judged mutagenic.
- A chemical was judged mutagenic (+) or weakly mutagenic (+ W) if it produced a reproducible dose-related response over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials.
- Chemicals were judged non-mutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or non-mutagenicity.

Key result

Species / strain:

S. typhimurium, other: TA97, TA98, TA100, TA1535, and TA1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The test material was reported to be mutagenic to the Salmonella typhimunum strains TA97, TA98, TA100, TA1535, and TA1537.

Executive summary:

The genetic toxicity of the test material was investigated in a study that followed similar principles to the guideline OECD 471.

The test material was examined using the pre-incubation assay, both in the presence and absence of metabolic activation in the form of S9 fractions. At least five doses of the test material were tested in triplicate.

The test material was reported to be mutagenic to the Salmonella typhimunum strains TA97, TA98, TA100, TA1535, and TA1537.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus Tests SHG 182/79184 and SHG 191 -79738

(Read-Across Study performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))

In both mouse micronuclues tests, it was concluded that the test material failed to show any evidence of mutagenic potential, when administered orally. However, at the higher dose levels evidence of toxicity, as shown by bone marrow depression, was observed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Type of assay:

other: mammalian erythrocyte micronucleus test (migrated information)

Species:

mouse

Strain:

other: CFLP

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 19 to 23 g
- Assigned to test groups randomly: yes
- Fasting period before study: animals were starved overnight before dosing
- Housing: each group of 5 mice were kept in a plastic disposable cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 50 ± 5 %
- Air changes (per hr): 20
- Photoperiod: The room was illuminated by artificial light for 12 hours per day

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Dosing volume: 0.1 mL/10 g bw per dose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Preparation: The formulations were prepared using a Silverson high speed mixer.

Duration of treatment / exposure:

The total doses were given as two equal administrations separated by an interval of 24 hours.

Frequency of treatment:

Two equal administrations separated by an interval of 24 hours.

Post exposure period:

6 hours following the second dose.

Dose / conc.:

2 250 mg/kg bw (total dose)

Dose / conc.:

4 500 mg/kg bw (total dose)

Dose / conc.:

9 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Phase I (preliminary toxicity study): 2 animals per sex per dose
Phase II (micronucleus study): 5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- Mitomycin C
- Doses / concentrations: Prepared at 0.7 mg/mL for two doses of 7 mg/kg, in 0.9 % saline
- Route of administration: Intra-peritoneally

Tissues and cell types examined:

- The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic and 2000 normochromatic erythrocytes per animal.
- In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- Doses for the micronucleus assay were selected from the results of the preliminary toxicity study.

DETAILS OF SLIDE PREPARATION:
- Following the last dose, the animals were observed for a further six hours before killing and all mortalities and signs of malreaction during the experiment were recorded. The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and one epiphysis removed from each bone.
- A direct bone marrow smear was made onto a slide containing a drop of calf serum. The slide was cleaned by immersion in methanol for 24 hours and air-dried immediately before use. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol overnight.
- After fixation, the smears were air-dried and placed in buffered distilled water (pH 6.8) for 10 minutes prior to staining in Giemsa (dilution factor 1 part Giemsa: 9 parts buffered distilled water pH 6.8) for 10 minutes. After rinsing in buffered distilled water (pH 6.8), the slides were air-dried. Prior to mounting in DPX, the slides were defatted by immersion in xylene for 10 minutes.

METHOD OF ANALYSIS:
- The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic and 2000 normochromatic erythrocytes per animal. In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal.

Statistics:

STATISTICAL ANALYSIS OF THE RATIOS OF NORMOCHROMATIC TO POLYCHROMATIC ERYTHROCYTES
- Due to heterogeneity of variance (Barlett's test; P < 0.001) non-parametric methods based on Kruskal-Wallis mean ranks were used to analyse the ratios of normochromatic to polychromatic erythrocytes. These methods have been found to be more robust against inequality of variance than classical analysis of variance methods.
- The positive control group dosed with Mitomycin C and the groups dosed with the test material were compared with the vehicle control using the non-parametric equivalent of the method of L.S.D.'s (least significant differences).
- After administration of the test material at a total dosage of 2250 mg/kg bodyweight, the ratio of normochromatic to polychromatic erythrocytes was not significantly different from the concurrent control value. At total dosages of 4500 and 9000 mg/kg body weight the ratios were significantly different from the concurrent control value, P < 0.01 and P < 0.001 respectively
- The positive control group dosed with Mitomycin C, gave a ratio which was significantly different from the concurrent control value, P < 0.001.

STATISTICAL ANALYSIS OF THE MICRONUCLEATED NORMOCHROMATIC AND POLYCHROMATIC ERYTHROCYTE COUNTS
- The non-parametric methods of statistical analysis detailed above were used to analyse the micronucleated normochromatic and polychromatic erythrocyte counts.
- After administration of the test material at all dosages, the micronucleated normochromatic and polychromatic erythrocyte counts were not significantly different from the respective concurrent control values.
- The positive control group dosed with Mitomycin C gave micronucleated cell counts which were significantly different from the respective concurrent control values, P < 0.001

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Bone marrow depression

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 4000, 8000, 9000, 10000, 11000, 12000, 14000 and 20000 mg/kg bw
- Clinical signs of toxicity in test animals: No toxic reactions were observed at 2000, 4000, 8000 and 9000 mg/kg bw. At 10000 and 11000 mg/kg bw, a toxic reaction consisting of hypopnoea was observed. At total dosages of 12000, 14000 and 20000 mg/kg bw, a toxic reaction consisting of hypopnoea and a period of lethargy was observed.
- From the above results, a top dosage of 9000 mg/kg bodyweight was chosen for the micronucleus test which, it was indicated, would cause one or two deaths.

RESULTS OF DEFINITIVE STUDY

SIGNS AND MORTALITIES
- After administration of 1 % methylcellulose, the vehicle control, no toxic reactions or mortalities were observed.
- After administration of the test material at all dosages, no toxic reactions were observed. At a total dosage of 9000 mg/kg bodyweight, there were three mortalities. Two animals died overnight after the administration of the first dose, between 8 and 24 hours. Advanced visceral autolysis prevented any post-mortem examination. The third died after administration of the second dose between 24 and 30 hours. Macroscopic examination at post-mortem failed to reveal any abnormalities.
- After administration of Mitomycin C, the positive control compound, no toxic reactions or mortalities were observed.

MICRONUCLEUS COUNTS
- The group mean counts and ranges obtained with the test material are shown in Table 1.
- In this experiment the negative control group gave a mean count of 2.8 micronucleated polychromatic cells which was just outside the range for previous unrelated experiments. However, the individual counts were within the laboratory standard range of individual counts for negative controls obtained in previous unrelated experiments.
- After administration of the test material at all dosages, the group mean polychromatic cell counts were comparable with the concurrent control value and within the laboratory standard range for negative controls obtained in previous unrelated experiments. In addition, the group mean micronucleated normochromatic cell counts of the treatment groups were comparable with the concurrent control value.
- The positive control, Mitomycin C, administered intraperitoneally gave a mean count of 89.5 micronucleated cells per 2000 polychromatic erythrocytes. This group also gave a mean count of 11.4 micronucleated cells per 2000 normochromatic erythrocytes.

Table 1: Group mean number of micronucleated cells per 2000 normochromatic erythrocytes and 2000 polychromatic erythrocytes per animal and the group mean ratio of normochromatic to polychromatic erythrocytes

Treatment	Total dosage over 24 h (mg/kg)	Number of micronucleated cells per 2000 erythrocytes per animal				Ratio of normochromatic to polychromatic erythrocytes
		Normochromatic		Polychromatic
		Mean	Range	Mean	Range	Mean	Range
Vehicle	-	2.5	0 to 6	2.8	1 to 6	1.09	0.93 to 1.22
Test material	2250	2.3	0 to 6	2.5	0 to 5	1.38	1.19 to 1.69
	4500	2.4	0 to 5	2.4	0 to 6	1.72**	1.47 to 2.09
	9000	2.3	0 to 6	2.6	1 to 5	3.93***	2.56 to 5.39
Positive control	14	11.4***	8 to 15	89.5***	74 to 115	10.46***	7.75 to 15.38

** P < 0.01

*** P < 0.001

Conclusions:

Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential, when administered orally. However, at the higher dose levels evidence of toxicity, as shown by bone marrow depression, was observed.

Executive summary:

A study was performed to investigate the genetic toxicity in vivo of the test material. The study was conducted in a style similar to OECD 474.

In this assessment of the effect of the test material on the incidence of micronucleated polychromatic erythrocytes in mice, total dosages of 2250, 4500 and 9000 mg/kg bodyweight were administered by oral gavage, in two equal dosages, separated by an interval of 24 hours.

A negative control group was dosed in an identical manner with the vehicle, 1 % methylcellulose. A positive control group was dosed by intraperitoneal injection with Mitomycin C, at a total dosage of 14 mg/kg bodyweight. The mice were sacrificed six hours after the second dose and bone marrow smears examined for the presence of micronuclei in 2000 polychromatic and 2000 normochromatic erythrocytes per mouse. The ratio of normochromatic to polychromatic cells was also examined in each mouse.

A preliminary toxicity study indicated that a top dosage of 9000 mg/kg bodyweight would cause one or two deaths and led to the selection of the dose concentrations for the main test.

At all dosages of the test material both of the group mean micronucleated cell counts were comparable with the concurrent control values.

After administration of the test material at a total dosage of 2250 mg/kg bodyweight, the ratio of normochromatic to polychromatic cells was comparable with the concurrent control value. At total dosages of 4500 and 9000 mg/kg bodyweight, the ratios were higher than the concurrent control value. Statistical analysis, using Kruskal-Wallis methods, showed these increases to be significantly different. The positive control compound, Mitomycin C, produced the expected large increase in the group mean polychromatic micronucleated cell count and in the normochromatic to polychromatic cell ratio. This group also produced a small increase in the group mean micronucleated normochromatic cell count.

Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential, when administered orally. However, at the higher dose levels evidence of toxicity, as shown by bone marrow depression, was observed.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

not specified

Type of assay:

other: mammalian erythrocyte micronucleus test (migrated information)

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 23 - 25 g
- Assigned to test groups randomly: yes
- Fasting period before study: animals were starved overnight prior to dosing
- Housing: groups of 5 mice in plastic disposable cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 5 %
- Air changes: 20 per hour
- Photoperiod: illuminated by artificial light for 12 hours per day

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1 % Methyl cellulose
- Amount of vehicle: 0.1 mL/ 10 g bw per dose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test material was prepared in the vehicle using a Silverson high speed mixer

Duration of treatment / exposure:

- The total doses were given as two equal administrations separated by an interval of 24 hours.

Frequency of treatment:

- Two equal administrations separated by an interval of 24 hours.

Post exposure period:

- Five male and five female mice from each dosage group were killed by cervical dislocation, at intervals of 12, 24, 36 and 48 hours after the last dose.

Dose / conc.:

4 500 mg/kg bw (total dose)

No. of animals per sex per dose:

20 animals per sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- Methotrexate
- Route of administration: intraperitoneal injection
- Doses / concentrations: 40 mg/kg (total dose)
- Vehicle: 0.9 % saline

Tissues and cell types examined:

- The incidence of micronucleated cells per 2000 normochromatic and 2000 polychromatic erythrocytes per animalwas determined.
- In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dosage of test material used in this study was the intermediate dose level used in a previous experiment.

TREATMENT AND SAMPLING TIMES: The chosen total dosage of 4500 mg/kg bodyweight was given as two equal administrations separated by an interval of 24 hours. The mice were starved overnight prior to dosing with the standard volume of 0.1 mL/10 g bodyweight per dose.

DETAILS OF SLIDE PREPARATION:
- The femurs were cleared of tissue and one epiphysis removed. Direct bone marrow smears were made onto slides containing a drop of calf serum.
- The slides were cleaned by immersion in methanol for 24 hours and air-dried immediately before use. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol overnight. After fixation, the smears were air-dried and placed in buffered distilled water (pH 6.8) for 10 minutes prior to staining in Giemsa (dilution factor 1 part Giemsa: 9 parts buffered distilled water pH 6.8) for 10 minutes. After rinsing in buffered distilled water (pH 6.8), the slides were air-dried.
- Prior to mounting in DPX, the slides were defatted by immersion in xylene for 10 minutes.

METHOD OF ANALYSIS: The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 normochromatic and 2000 polychromatic erythrocytes per animal. In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal.

Statistics:

Statistical analysis of the micronucleated normochromatic and polychromatic erythrocyte counts:
- Due to heterogeneity of variance (Bartlett's test; P < 0.001) non-parametric methods based on Kruskal-Wallis mean ranks were used to analyse all the data from this experiment. These methods have been found to be more robust against inequality of variance than classical analysis of variance methods.

Statistical analysis of the ratios of normochromatic to polychromatic erythrocytes:
- The non-parametric methods of statistical analysis detailed above were used to analyse the ratio of normochromatic to polychromatic erythrocytes.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY

Clinical signs and mortalities:
- After administration of 1 % Methylcellulose, the vehicle control, no toxic reactions or mortalities were observed.
- After administration of the test material no toxic reactions were observed until 36 hours after the second dose when a reaction consisting of a period of lethargy and ptosis was observed. At 48 hours after the second dose an additional toxic reaction consisting of hypopnoea and tremors was observed. There were four mortalities in this group, animals died overnight between 36 and 48 hours.
- After administration of the positive control compound, no toxic reactions or mortalities were observed.

Induction of micronuclei:
- In this experiment the negative control group gave mean counts of 2.4, 2.6, 2.6 and 2.3 micronucleated polychromatic erythrocytes at the 12, 24, 36 and 48 hour kills respectively. This group also gave mean counts of 2.0, 2.3, 2.0 and 2.0 micronucleated normochromatic erythrocytes at the 12, 24, 36 and 48 hour kills respectively.
- After administration of the test material the group mean micronucleated polychromatic cell counts were comparable with the concurrent control value at the four periods tested. The group mean micronucleated normochromatic erythrocyte counts at the 12, 24 and 36 hour kills were also comparable with the concurrent control value. After 48 hours however the group mean micronucleated normochromatic cell count was slightly increased over the concurrent control value.
- The positive control, administered intraperitoneally, gave group mean counts of 14.2, 19.6 and 42.3 micronucleated polychromatic cells at the 12, 24 and 36 hour kills respectively. There were too few cells to count at the 48 hour kill. This group also gave mean counts of 11.3, 13.8 and 19.3 micronucleated normochromatic cells at the 12, 24 and 36 hour kills respectively. At the 48 hour kill the group mean count was 2.7 micronucleated normochromatic cells which was comparable with the concurrent control value.

Ratio of PCE/NCE (for Micronucleus assay):
- In this experiment, the negative control group, gave group mean ratios of 0.97, 1.01, 1.01 and 1.06 normochromatic to polychromatic cells, at the 12, 24, 36 and 48 hour kills respectively.
- After administration of the test material the group mean ratio at each period was higher than that obtained with the concurrent control. The ratio increased with time after administration of the second dose.
- The positive control, administered intraperitoneally, gave group mean ratios of 2.07, 10.76 and 19.18 normochromatic to polychromatic cells at the 12, 24 and 36 hour kills respectively. After 48 hours there were too few polychromatic cells to count and therefore no cell ratio value was recorded.

Statistical analysis of the micronucleated normochromatic and polychromatic erythrocyte counts:
- The positive control group and the groups dosed with the test material were compared with the vehicle control group using the non-parametric equivalent of the method of L.S.D.'s (least, significant differences).
- The positive control group and the vehicle control group were the same as those used in a previous report.
- After administration of the test material, the micronucleated normochromatic erthrocyte counts at the 12, 24 and 36 hour kills were not significantly different from the respective concurrent control values. At the 48 hour kill the micronucleated normochromatic cell count was significantly different from the concurrent control value P < 0.05. The micronucleated polychromatic erythrocyte counts at each kill were not significantly different from the respective concurrent control values.
- The positive control group gave micronucleated polychromatic and normochromatic cell counts at the 12, 24 and 36 hour kills which were significantly different from the respective concurrent control values P < 0.001. At the 48 hour kill the micronucleated normochromatic cell count was not significantly different from the concurrent control value.

Statistical analysis of the ratios of normochromatic to polychromatic erythrocytes:
- After administration of the test material the ratio of normochromatic to polychromatic erythrocytes were significantly different from the respective concurrent control values at each kill. At the 12 and 24 hour kills P < 0.05 and at the 36 and 48 hour kills P < 0.01.
- The positive control group, gave ratios at the 12, 24 and 36 hour kills which were significantly different from the respective concurrent control values, P < 0.001.

CONCLUSIONS

- From these results, it is concluded that the test material does not show any evidence of mutagenic potential when administered orally, in this test procedure. However evidence of bone marrow depression was observed in the smears from each kill.

- The small, but statistically significant, rise in the micronucleated normochromatic erythrocytes at the 48 hour kill cannot be taken as evidence of mutagenic potential due to anti-metabolite action. In the event of cell cycle delay a corresponding rise in micronucleated polychromatic cells would have been expected prior to an increase in micronucleated normochromatic cells. In this experiment the test material did not cause this rise in the micronucleated polychromatic cells and therefore the slight increase in the micronucleated normochromatic cells must remain an unexplained phenomenon. It is possible that it may be related to the high toxicity of the test material as shown by the degree of bone marrow depression.

- The positive control compound, gave the expected rises in the micronucleated normochromatic and polychromatic erythrocyte counts in mice killed 12, 24 and 36 hours after the second dose. In mice killed 48 hours after the second dose there were too few polychromatic erythrocytes to count. In addition, at the 48 hour kill, the value of the mean micronucleated normochromatic erythrocyte count dropped so as to become comparable with the concurrent control value. These two findings may be explained by the high level of toxicity to the bone marrow cells resulting in massive numbers of deaths in the cell population, so causing a reduction in the number of polychromatic erythrocytes and the consequent shunting of peripheral blood into the bone marrow to compensate for this. Hence the frequency of micronucleated normochromatic erythrocytes from the bone marrow exposed to Methotrexate would be reduced to control levels due to the influx of cells from the circulating blood.

Conclusions:

Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential when administered orally. However, evidence of toxicity as shown by bone marrow depression was observed.

Executive summary:

A study was performed to investigate the genetic toxicity in vivo of the test material. The study was conducted in a style similar to OECD 474 using the micronucleus test on male and female CD-1 mice.

The chosen total dosage of 4500 mg/kg bodyweight (the intermediate dose level from a previous study) was given as two equal administrations separated by an interval of 24 hours. The mice were starved overnight prior to dosing with the standard volume of 0.1 mL/10 g bodyweight per dose.

Following the last dose, the animals were observed and all mortalities and signs of malreaction during the experiment were recorded. Five male and five female mice from each dosage group were killed by cervical dislocation, at intervals of 12, 24, 36 and 48 hours after the last dose and both femurs dissected from each animal. Direct bone marrow smears were made onto slides containing a drop of calf serum. The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 normochromatic and 2000 polychromatic erythrocytes per animal. In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal.

The small, but statistically significant, rise in the micronucleated normochromatic erythrocytes at the 48 hour kill cannot be taken as evidence of mutagenic potential due to anti-metabolite action. In the event of cell cycle delay a corresponding rise in micronucleated polychromatic cells would have been expected prior to an increase in micronucleated normochromatic cells. In this experiment the test material did not cause this rise in the micronucleated polychromatic cells and therefore the slight increase in the micronucleated normochromatic cells must remain an unexplained phenomenon. It is possible that it may be related to the high toxicity of the test material as shown by the degree of bone marrow depression.

The positive and negative controls used in the study were judged to be valid.

Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential when administered orally. However, evidence of toxicity as shown by bone marrow depression was observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

IN VITRO

Gene Mutation study in Bacteria

Jagannath 1978 (Read-Across Study performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))

The genetic toxicity of the test material was investigated in a bacterial reverse mutation assay, broadly similar to OECD 471. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested at 5.000, 1.000, 0.100, 0.010, 0.001 µL/plate both with and without metabolic activation, in the form of S9-mix. The following bacterial strains were used: Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA1538, and Saccharomyces cerevisiae strain D4. The overlay method of application was used.

The plates were incubated for 48 hours at 37 °C, and scored for the number of colonies growing on each plate. Positive and solvent controls using both directly active positive chemicals and those that require metabolic activation were run with each assay. The numbers of colonies on each plate were counted and recorded.

The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The results of the tests conducted on the test material in the absence of a metabolic system were all negative. The results of the tests conducted on the test material in the presence of the rat liver activation system were all negative.

Under the conditions of the study, the test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic with or without metabolic activation.

Gene Mutation study in Bacteria

Gunzel 1979 (Read-Across Study performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))

The 1978 (Jagannath) study was repeated with TA100 at higher test material concentrations due to the slightly increased (and dose-dependent) number of mutants of Salmonella typhimurium TA 100 in the highest tested concentration in the absence of metabolic activation.

The results of the tests conducted on the test material in the absence of a metabolic system were all negative. The results of the tests conducted on the test material in the presence of the rat liver activation system were all negative.

Under the conditions of this study the test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic under these test conditions.

Gene Mutation study in Bacteria

Zieger 1988 (Read-Across Study performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))

The genetic toxicity of the test material was investigated in a study that followed similar principles to the guideline OECD 471.

The test material was examined using the pre-incubation assay, both in the presence and absence of metabolic activation in the form of S9 fractions. At least five doses of the test material were tested in triplicate.

The test material was reported to be mutagenic to the Salmonella typhimunum strains TA97, TA98, TA100, TA1535, and TA1537.

Gene Mutation study in Bacteria

Read-across data (mixture DOTE: MOTE. 70:30) (CAS No 15571-58-1 and CAS 27107-89-7)

- Anonymous (1979): An Ames test was carried out with a mixture of 70% dioctyltin bis(2-ethylhexylmercaptoacetate) and 30% mono-octyltin tris(2-ethylhexylmercaptoacetate). The relatively old test was considered less reliable, because the test was not conducted using an E. coli strain and no positive controls for strains TA98, TA100, and TA1537 were included in the test with metabolic activation. No mutagenic activity was observed.

-Anonymous (1978) Two studies: In these studies the test material was positive in strain TA100 without S9. The other strains were negative.

- Anonmymous (1983): This Ames test was considered less reliable due to the fact that the test was not conducted using E. coli strain WP2 uvrA. Positive controls for the strains TA98, TA1537, and TA1538 were not included in the test with metabolic activation. The test material precipitated in the soft agar at concentrations of 8100 and 24,300 µg/0.1 mL. In the tests conducted without microsomal activation, there was a slight increase in the number of back-mutant colonies of strain TA100 in the 2700 µg/0.1 mL concentration and above. Additionally, a growth-inhibiting effect was reported in the tests without activation for strains TA1535, TA1537, and TA1538 at a concentration of 24,300 µg/0.1 mL. In the first assay with microsomal activation, there was a slight increase in the number of back-mutant colonies of strain TA1537 at 300 and 2700 µg/0.1 mL. The study authors attributed this to fluctuation in the rate of spontaneously occurring back-mutants. Negative and positive controls produced the expected mutant colony counts.

Mouse Lymphoma Assay Read-across to (DOTE) (CAS No 15571 -58 -1).

The test material DOTE was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). Three tests were conducted; in the first and third test single cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively; in the second test cultures were treated for 24 hours and 4 hours in the absence of S9-mix and 4 hours in the presence of S9-mix. The test material was dissolved in ethanol prior to testing.

The highest concentrations of DOTE evaluated for mutagenicity in the absence of S9-mix, were 20 μg/mL and 0.36 μg/mL at 4 and 24 hours exposure, respectively. In the presence of S9-mix and 4 hours exposure, the highest concentration evaluated was 85 μg/mL. The maximum concentrations were limited by cytotoxicity.

DOTE was cytotoxic in both the absence and presence of S9-mix. In the absence of S9-mix and 24 hours exposure cytotoxicity, resulting in a reduction in initial cell yield and/or suspension growth, was observed at and above 0.1 μg/mL. The relative total growth (RTG) at the highest concentration evaluated in the first and third test were 22 and 13 %, respectively. In the absence of S9-mix and 4 hours exposure cytotoxicity was observed at all concentrations included (as from 0.86 μg/mL). The relative total growth (RTG) at the highest concentration evaluated in the second test was 20 %.

In the presence of S9-mix cytotoxicity was observed at and above 1.4 μg/mL. The relative total growth (RTG) at the highest concentration evaluated in the first and third test were 38 and 30 %, respectively. In the absence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. In the presence of S9-mix at 72 μg/mL the mutant frequency was significantly increased by 238 mutants per 1,000,000 clonable cells compared to the negative control. Since relatively small intervals (0.85) were used and the increase was observed at a single concentration causing more than 90 % cytotoxicity compared to six concentrations causing 50-70 % cytotoxicity which showed no increase in mutant frequency, it is concluded that this increase is not indicative for mutagenicity. It is very likely a secondary effect to cytotoxicity and thus not indicative for mutagenicity.

Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and presence of the S9-mix, respectively; ethanol (vehicle) served as negative control. The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls.

It is concluded that under the conditions used in this study, the test material DOTE is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.

IN VIVO

Mouse Micronucleus Test 1980, SHG 182/79184 (Read-Across Study performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))

A study was performed to investigate the genetic toxicity in vivo of the test material. The study was conducted in a style similar to OECD 474. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

In this assessment of the effect of the test material on the incidence of micronucleated polychromatic erythrocytes in mice, total dosages of 2250, 4500 and 9000 mg/kg bodyweight were administered by oral gavage, in two equal dosages, separated by an interval of 24 hours.

A negative control group was dosed in an identical manner with the vehicle, 1 % methylcellulose. A positive control group was dosed by intraperitoneal injection with Mitomycin C, at a total dosage of 14 mg/kg bodyweight. The mice were sacrificed six hours after the second dose and bone marrow smears examined for the presence of micronuclei in 2000 polychromatic and 2000 normochromatic erythrocytes per mouse. The ratio of normochromatic to polychromatic cells was also examined in each mouse.

A preliminary toxicity study indicated that a top dosage of 9000 mg/kg bodyweight would cause one or two deaths and led to the selection of the dose concentrations for the main test.

At all dosages of the test material both of the group mean micronucleated cell counts were comparable with the concurrent control values.

After administration of the test material at a total dosage of 2250 mg/kg bodyweight, the ratio of normochromatic to polychromatic cells was comparable with the concurrent control value. At total dosages of 4500 and 9000 mg/kg bodyweight, the ratios were higher than the concurrent control value. Statistical analysis, using Kruskal-Wallis methods, showed these increases to be significantly different. The positive control compound, Mitomycin C, produced the expected large increase in the group mean polychromatic micronucleated cell count and in the normochromatic to polychromatic cell ratio. This group also produced a small increase in the group mean micronucleated normochromatic cell count.

Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential, when administered orally. However, at the higher dose levels evidence of toxicity, as shown by bone marrow depression, was observed.

Mouse Micronucleus Test 1980, SHG 191/79738 (Read-Across Study performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))

A study was performed to investigate the genetic toxicity in vivo of the test material. The study was conducted in a style similar to OECD 474 using the micronucleus test on male and female CD-1 mice. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The chosen total dosage of 4500 mg/kg bodyweight (the intermediate dose level from a previous study) was given as two equal administrations separated by an interval of 24 hours. The mice were starved overnight prior to dosing with the standard volume of 0.1 mL/10 g bodyweight per dose.

Following the last dose, the animals were observed and all mortalities and signs of malreaction during the experiment were recorded. Five male and five female mice from each dosage group were killed by cervical dislocation, at intervals of 12, 24, 36 and 48 hours after the last dose and both femurs dissected from each animal. Direct bone marrow smears were made onto slides containing a drop of calf serum. The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 normochromatic and 2000 polychromatic erythrocytes per animal. In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal.

The small, but statistically significant, rise in the micronucleated normochromatic erythrocytes at the 48 hour kill cannot be taken as evidence of mutagenic potential due to anti-metabolite action. In the event of cell cycle delay a corresponding rise in micronucleated polychromatic cells would have been expected prior to an increase in micronucleated normochromatic cells. In this experiment the test material did not cause this rise in the micronucleated polychromatic cells and therefore the slight increase in the micronucleated normochromatic cells must remain an unexplained phenomenon. It is possible that it may be related to the high toxicity of the test material as shown by the degree of bone marrow depression.

The positive and negative controls used in the study were judged to be valid.

Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential when administered orally. However, evidence of toxicity as shown by bone marrow depression was observed.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.