Tetrakis(diethyldithiocarbamato-S,S')tellurium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance scored negative in Ames tests similar to OECD 471. E. coli or S. typhimurium TA 102 tester strain were not tested.

The substance scored positive without S9 mix in the CA test similar to OECD 473.

The substance scored positive in the MLA test similar to OECD 490.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Ames test without E. coli or S. typhimurium TA 102 tester strain

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of the test item (as cited in study report): Tellurium diethyldithiocarbamate
- Batch No.: M7-5D-494
- Appearance: Yellow, powder

Target gene:

his

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate S-9 mix (Aroclor 1254 induced)

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150 µg/plate
The test substance was toxic towards the tester strains at the higher dose levels in the range finding test (5-5000 µg/plate). Therefore, 150 µg/plate was chosen as the top dose level in the mutation test.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

The following procedure is carried out on each bacterial strain:
Four concentrations of test substance are assessed for toxicity using the five tester strains. The highest concentration is usually 0.05 g of test substance dissolved in 1 ml of solvent. Three 10-fold serial dilutions of the top concentration are also tested. The chosen solvent is used as the negative control.

0.1 ml of an overnight bacterial culture containing approximately 2 x 10^9 cells/ml, and 0.5 ml S-9 mix or 0.5 ml 0.1 M sodium phosphate buffer (pH 7.4) are placed in glass bijou bottles. 0.1 ml of the test solution is added followed by 2 ml histidine deficient agar. The mixture is thoroughly shaken and overlaid onto previously prepared plates containing 15 ml minimal agar. Single petri dishes are used for each dose level. They are incubated at 37 °C for 72 hours. After this period the plates are examined for the appearance of a complete bacterial lawn. Revertant colonies are counted using a Biotran Automatic Colony Counter. Any toxic effects of the test substance are detected by a substantiai reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group.
A compound is deemed to provide evidence of mutagenic potential if (1) a statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments, and (2) the increase in the number of revertant colonies is at least twice the concurrent solvent control value.

Statistics:

Not specified

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537 and TA1538, TA98 and TA 100,

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance at any dose level, either in the presence or absence of metabolic activation (S-9 mix).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Description of methods is limited; Ames test without E. coli or S. typhimurium TA102 tester strain

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Ames test without E. coli or S. typhimurium TA102 tester strain

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name as cited in report: Ethyl Tellurac

Target gene:

his

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

induced male Sprague Dawley rat liver S9 or induced male Syrian hamster liver S9

Test concentrations with justification for top dose:

JUSTIFICATION
Top dose limited by cytotoxicity

TEST 1
- Without S9: 0.001, 0.003, 0.01, 0.03, 0.1 µg/plate
- With rat or hamster S9: 3.3, 10, 33, 100, 220 µg/plate

TEST 2
- Without S9: 0.001, 0.003, 0.01, 0.03 µg/plate
- With rat or hamster S9: 1, 3.3, 10, 33, 100 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See table in 'Any other information on materials and methods inc. tables'

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: not specified

METHOD
- Test solution (0.05 mL) was incubated with 0.05 or 0.1 mL of overnight culture plus 0.5 mL of hamster or rat S9 for 20 minutes, then top agar was added and the cells were plated on petri dishes containing Vogel-Bonner medium. The plates are then incubated (usually for two days).
- Plates were machine counted unless precipitate or colour interfered.

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

- The test is only valid if the number of positive control colonies is greater than for the control cultures. Once this has been confirmed, the test cultures are compared to the control. If the substance is mutagenic, it will have caused greater bacteria growth.
- A positive response is a reproducible, dose-related increase in any set of test cultures. There is no minimum percentage of increase to define a result as positive.
- An equivocal response is any increase that is not reproducible, not dose-related, or not statistically significant.
- A negative response occurs when none of the cultures tested shows more growth than the control.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was found

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- 220 µg/plate (with S9 mix) was toxic in all strains
- 0.03 and 0.1 µg/plate (without S9 mix) was slightly toxic in all strains, however not in both replicates

Tables. Overview results

Strain: TA100
Dose	No Activation		No Activation		10% RLI		10% RLI		10% HLI		10% HLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	t		145	2.3	131	5	118	11	121	6.4	115	11.3
0.001	117	10.2	137	8.4
0.003	126	9.3	155	6.4
0.01	134	6.7	159	3.2
0.03	105 s	.9	143 s	11.2
0	t		145	2.3	131	5	118	11	121	6.4	115	11.3
0.1	79 s	4.5
1							132	.9			103	3
3.3					106	8.2	134	7.5	131	6.1	95	5
10					135	7.1	137	9.1	132	2.3	112	5.8
33					130	.6	157	3.7	138	5.2	131	4.5
100					102	8.8	151	3.4	133	6.2	124	9.3
220					t				t
Positive Control	2063	27.7	2171	43	1221	26.6	1232	94.3	2346	65.7	2418	120.4

 

Strain: TA1535
Dose	No Activation		No Activation		10% RLI		10% RLI		10% HLI		10% HLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	26	1.7	31	1.2	11	1.7	15	2.2	13	.3	15	.7
0.001	19	.6	39	1.7
0.003	27	3.1	43	4.5
0.01	28	.9	37	6.5
0.03	27 s	.6	42	4.7
0	26	1.7	31	1.2	11	1.7	15	2.2	13	.3	15	.7
0.1	15 s	.6
1							13	1.2			19	.9
3.3					9	.7	14	1.5	11	.7	13	3.2
10					11	3.5	12	.3	7	0	8	2.2
33					7	1.2	14	3	10	1.7	13	2.6
100					8	.6	20	3.4	9	1.2	18	2.3
220					t				t
Positive Control	1377	48.5	1347	9.7	54	1.8	128	10.6	110	3.4	195	3.8

 

Strain: TA1537
Dose	No Activation		No Activation		10% RLI		10% RLI		10% HLI		10% HLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	8	.6	8	1.2	11	2.7	6	2.1	8	1.2	8	.9
0.001	8	.9	6	.9
0.003	9	.6	9	.7
0.01	7	3.6	11	1.5
0.03	9 s	1.8	4	2.1
0	8	.6	8	1.2	11	2.7	6	2.1	8	1.2	8	.9
0.1	7 s	1.8
1							9	1			9	1.7
3.3					11	2.6	6	2.6	6	1	11	3.3
10					5	.6	9	3	11	.9	11	.9
33					11	1.5	9	.7	9	1.3	11	1.2
100					8	1.5	10	.9	9	4.4	12	1.5
220					t				t
Positive Control	3593 s	262.1	926	65	95	10.5	71	5.7	220	16.7	262	13.1

 

Strain: TA98
Dose	No Activation		No Activation		10% RLI		10% RLI		10% HLI		10% HLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	18	3.3	18	2.4	24	.9	24	1	23	.9	29	5.5
0.001	19	2.9	17	.3
0.003	14	5	20	1.7
0.01	12	.7	21	3.4
0.03	13 s	1.8	19 s	3.1
0	18	3.3	18	2.4	24	.9	24	1	23	.9	29	5.5
0.1	12 s	.3
1							28	2.7			37	8.7
3.3					27	2.5	26	3.8	24	1	29	2.8
10					28	3.5	26	2	29	2	32	2.3
33					24	3.8	24	1.2	26	4	29	.9
100					14	2.4	18	2.6	10	.9	21	.3
220					t				t
Positive Control	2461	26.2	1904	23.5	981	14.2	1053	17.1	2161	57.2	1932	50.4

 

Abbreviations:

RLI = induced male Sprague Dawley rat liver S9

HLI = induced male Syrian hamster liver S9

s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; t = Toxic;

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Description of methods is limited; Ames test without E. coli or S. typhimurium TA102 tester strain

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Ames test without E. coli or S. typhimurium TA102 tester strain

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name as cited in report: Ethyl Tellurac

Target gene:

his

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

induced male Sprague Dawley rat liver S9 or induced male Syrian hamster liver S9

Test concentrations with justification for top dose:

JUSTIFICATION
Top dose without S9 limited by cytotoxicity

TEST 1
- Without S9: 0.001, 0.33, 1.0, 3.3, 10.0, 33 µg/plate
- With rat or hamster S9: 0.33, 1.0, 3.3, 10.0, 33.0 µg/plate

TEST 2
- Without S9: 0.033, 0.33, 0.1, 1.0, 3.3 µg/plate
- With rat or hamster S9: 0.33, 1.0, 3.3, 10.0, 33.0 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See table in 'Any other information on materials and methods inc. tables'

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: not specified

METHOD
- Test solution (0.05 mL) was incubated with 0.05 or 0.1 mL of overnight culture plus 0.5 mL of hamster or rat S9 for 20 minutes, then top agar was added and the cells were plated on petri dishes containing Vogel-Bonner medium. The plates are then incubated (usually for two days).
- Plates were machine counted unless precipitate or colour interfered.

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

- The test is only valid if the number of positive control colonies is greater than for the control cultures. Once this has been confirmed, the test cultures are compared to the control. If the substance is mutagenic, it will have caused greater bacteria growth.
- A positive response is a reproducible, dose-related increase in any set of test cultures. There is no minimum percentage of increase to define a result as positive.
- An equivocal response is any increase that is not reproducible, not dose-related, or not statistically significant.
- A negative response occurs when none of the cultures tested shows more growth than the control.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was found

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- All concentrations were nontoxic in presence of S9 mix
- Toxicity was observed without S9 mix, at concentrations of 3.3 µg/plate and higher (TA100, TA1535, TA1537) or 10 µg/plate and higher (TA98)

Tables. Overview results

 

Strain: TA100
Dose	No Activation		No Activation		10% RLI		10% RLI		10% HLI		10% HLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	167	11.6	111	17.8	235	11	111	3.6	227	14.3	113	.6
0.033			87	7.8
0.33	147	3	88	11.3	263	18.3	129	3.2	255	9.6	114	4.7
0.1			82	4
1	161	11.1	88	16.9	270	8.7	119	3.4	263	5.9	119	3.8
3.3	t		72	2.8	248	8	109	4.7	221	1.5	125	12.2
10	t				267	24.3	122	8.4	253	4.3	110	10.2
33	t				305	8.7	115	9.8	227	2.8	120	15.4
Positive Control	634	40.5	426	54.5	1322	58.6	688	41.9	3105	20.3	2007	110.3

 

Strain: TA1535
Dose	No Activation		No Activation		10% RLI		10% RLI		10% HLI		10% HLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	6	.3	6	.6	6	.6	9	1.8	8	.9	14	3.5
0.33	12	4.5	10	.7	6	.6	13	2.1	6	1	12	1
0.1			7	1.5
1	4	.3	2	1	6	.3	10	.6	7	1.2	13	1.2
3.3	4	.3	t		7	1.2	15	2.6	8	.7	15	.9
10	t		t		5	.3	9	1.3	7	1.7	11	.7
33	t				5	.3	11	2.7	8	1.3	11	2.2
Positive Control	834	35.9	617	23	137	11.5	56	8.7	319	39	93	4.2

 

Strain: TA1537
Dose	No Activation		No Activation		10% RLI		10% RLI		10% HLI		10% HLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	4	.9	5	.7	13	2.1	7	.9	11	.3	6	2.2
0.033			3	.9
0.33	3	.6	2	1.2	15	2.5	9	.3	4	.3	4	1.9
0.1			3	1.5
1	3	.7	t		23	3	6	3.3	4	.6	5	1.5
3.3	t		t		13	1.9	4	1.5	8	1.7	4	.9
10	t				14	1.8	6	1.2	8	.9	5	.7
33	t				14	1.5	5	1.8	11	1.5	7	1.3
Positive Control	697	43.2	662	65.2	212	28.9	31	2.3	688	63.8	70	3.8

 

Strain: TA98
Dose	No Activation		No Activation		10% RLI		10% RLI		10% HLI		10% HLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	17	3.3	13	1.9	25	1.5	18	3.5	24	2.4	21	1.7
0.33	13	2.6	7	1.7	23	3.5	21	2.2	23	.6	24	2.1
1	10	2	7	.7	27	0	20	1.3	21	1	24	3.8
3.3	5	0	6	1.2	17	.3	21	4.6	26	2.8	25	1.2
10	2	0	t		15	2.6	15	2.1	15	2	14	1.8
33	1	.3	t		18	2.2	25	6.1	24	2.1	15	3.2
Positive Control	339	14.6	144	11.7	536	77.7	318	35.8	2249	112.8	1836	16.1

 

Abbreviations:

RLI = induced male Sprague Dawley rat liver S9

HLI = induced male Syrian hamster liver S9

s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; t = Toxic;

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

The test was performed according to the NTP protocol for Chinese Hamster Ovary Cell Cytogenetics (CA test). This protocol has been published (Galloway et al., 1985; PMID 3967632)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name as cited in report: Ethyl Tellurac
- Source of test material: Radian Corporation

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-W-B1

Details on mammalian cell type (if applicable):

CELLS USED
- Methods for maintenance in cell culture: 5% CO2, maximum 37°C

MEDIA USED
- Type and identity of media: McCoy's 5A medium supplemented with fetal calf serum (10%), L-glutamine (2mM), and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin)

Cytokinesis block (if used):

Colcemid

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver S9

Test concentrations with justification for top dose:

JUSTIFICATION
The dose levels were determined from the information on cell cycling and toxicity obtained in a CA test.

DOSES
- without S9: 0.032, 0.1, 0.15, 0.32 µg/mL
- with S9: 0.1, 0.32, 1.0, 15.0 µg/mL

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 8-15 hours (without S9) or 2 hours (with S9). Incubation time without S9 was determined from the information on cell cycling form the SCE test: if cell cycle delay was anticipated in the CA test, the incubation period was extended to permit accumulation of sufficient cells in first metaphase for analysis
- Expression time (cells in growth medium): 10 hours (only following incubation with S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 14 hours

SPINDLE INHIBITOR:
Colcemid, added during the final 2-3 hours of incubation

STAIN:
Giemsa

NUMBER OF REPLICATIONS:
1

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Two to three hours after addition of colcemid, cells were collected by mitotic shake-off and treated for up to 3 min at room temperature with hypotonic KCl (75 mM). Cells were then washed twice with fixative (3:1, methanol:glacial acetic acid, v/v), dropped onto slides, and air-dried

NUMBER OF CELLS EVALUATED:
100

Statistics:

Statistical analyses were performed to compare exposed cells to vehicle controls.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-W-B1

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-W-B1

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1. Overview results without S9 mix

	Dose µg/mL	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Aberrations
		No. of Abs	Abs Per Cell	% Cells with abs	No. of Abs	Abs Per Cell	% Cells with abs	No. of Abs	Abs Per Cell	% Cells with abs	No. of Abs	Abs Per Cell	% Cells with abs
Dimethyl Sulfoxide	0	3	0.03	3	0	0	0	3	0.03	3	0	0	0
Test chemical	0.032	10	0.1	8	2	0.02	2	8	0.08	6	0	0	0
Test chemical	0.1	26	0.26	19	0	0	0	26	0.26	19	0	0	0
Test chemical	0.32	15	0.15	11	2	0.02	2	13	0.13	9	0	0	0
Positive control: Mitomycin-C	0.15	42	0.42	29	14	0.14	11	28	0.28	22	0	0	0
Trend:		2.57			0.904			2.379
Probability:		0.005			0.183			0.009

Table 2. Overview results with S9 mix

	Dose µg/mL	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Aberrations
		No. of Abs	Abs Per Cell	% Cells with abs	No. of Abs	Abs Per Cell	% Cells with abs	No. of Abs	Abs Per Cell	% Cells with abs	No. of Abs	Abs Per Cell	% Cells with abs
Dimethyl Sulfoxide	0	4	0.04	4	1	0.01	1	3	0.03	3	0	0	0
Test chemical	0.1	4	0.04	3	1	0.01	1	3	0.03	2	0	0	0
Test chemical	0.32	6	0.06	6	2	0.02	2	4	0.04	4	0	0	0
Test chemical	1	6	0.06	6	2	0.02	2	4	0.04	4	0	0	0
Positive control: Mitomycin-C	0.15	19	0.38	30	7	0.14	14	12	0.24	20	0	0	0
Trend:		0.948			0.737			0.632
Probability:		0.172			0.231			0.264

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Description of methods is limited

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

yes

Remarks:

relative total growth is not reported

Principles of method if other than guideline:

The test was performed according to the NTP protocol for Mouse Lymphoma: Mammalian Cell Mutagenicity. This protocol has been published (Mitchell et al., 1988; PMID 3416837)

GLP compliance:

not specified

Remarks:

The year indicated in the box is the accessed year.

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name as cited in report: Ethyl Tellurac

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

3.7.2C

Details on mammalian cell type (if applicable):

CELLS USED
- cycling time: about 10 hours
- Methods for maintenance in cell culture: Cells were kept at 37° C as suspension cultures in the media described below

MEDIA USED
- Type and identity of media: Fischer's medium, with an additional 2 mM l-glutamine, 110 µg/mL sodium pyruvate, 0.05% luronic F68, antibiotics, and heat-inactivated horse serum.
- Periodically 'cleansed' against high spontaneous background: To limit random mutations, cultures were treated with thymidine, hypoxanthine, methotrexate, and glycine. Stock cultures were grown in F10p medium containing THMG (3 µg/mL thymidine, 5 µg/mL hypoxanthine, 0.1 µg/mL methotrexate and 7.5 µg/mL glycine) for 24 hours then moved to medium with THG for 1 to 3 days prior to use. Periodically frozen cells were used to start new cultures.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced or non-induced male Fisher 344 rat liver S9

Test concentrations with justification for top dose:

JUSTIFICATION
The highest concentration was chosen using cell toxicity, substance solubility, or a hard upper limit of 5 mg/mL.

CONCENTRATIONS
- without S9, trial 1: 1.25, 2.5, 5, 10, 20, 30, 40 µg/mL
- without S9, trial 2: 2.5, 5, 10, 20, 30, 40 µg/mL
- with S9, trial 1: 0.313, 0.625, 1.25, 2.5, 5, 10 µg/mL
- with S9, trial 2: 2.5, 5, 7.5, 10, 15 µg/mL

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: Each culture contained 6 x 10^6 cells and 10 mL of medium dosed with the test substance.

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): two days
- Selection time (if incubation with a selection agent): ten to twelve days

SELECTION AGENT:
trifluorothymidine

NUMBER OF REPLICATIONS:
3

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

DETAILED METHODS
- Treated cultures contained 6x10^6 cells in 10 mL of medium. This volume included the S9 fraction if metabolic activation was included.
- After exposure to test article the medium plus chemical was removed and the cells resuspended in 20 mL of fresh medium.
- After the 48- hour expression period, 3x10^6 cells were plated in medium and soft agar supplemented with trifluorothymidine for selection of TFT-resistant cells and in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 degrees in 5% CO2
- At the end of incubation, colonies were counted with an automatic counter.

Evaluation criteria:

For the test to be valid, control cultures and test cultures had to show cloning efficiency within a certain range. Each dose set had to produce at least two valid cultures. If the test chemical precipitated out of the medium, the data was thrown out.
- For a positive result, both cultures had to show significant induced TFT resistance.
- If one showed a significant response, the result was marked as questionable.
- If a clearly positive response was not obtained in activated cultures, the test was repeated using freshly prepared S9 from the livers of either Aroclor 1254-induced or non-induced male Fischer 344 rats.
- If there was no trend of increasing response and no peak, the test was negative.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

See 'any other information on results incl. tables'

Overview results

Nonactivation Trial: 1 Experiment Call: Positive

	Conc.	Cloning	Relative Total	Mutant Colonies	Mutant Frequency	Avg Mutant Frequency
	µg/mL	Efficiency	Growth
Vehicle Control (Dimethyl Sulfoxide)	0	141r	133	209	49	65
		104	98	181	58
		150r	114	248	55
		87	102	185	71
Test Chemical	1.25	101	94	143	47	49
		93	79	152	55
		108	94	143	44
	2.5	99	90	138	47	47
		83	96	124	50
		102	101	136	44
	5	115#	89	241	70	80
		101	90	269	89
		138r	123	161	39
	10	100	77	163	54	66
		73	77	173	79
		93	80	181	65
	20	105	44	191	61	70
		56	22	125	75
		69	21	156	76
	30	73	14	238	108	134*
		65	11	262	135
		61	6	289	157
	40
Positive Control (Ethyl Methane Sulfonate)	250	84#	55	1023	405	389*
		95	65	1071	377
		92	69	1063	384

 

Nonactivation Trial: 2 Experiment Call: Positive

	Conc.	Cloning	Relative Total	Mutant Colonies	Mutant Frequency	Avg Mutant Frequency
	µg/mL	Efficiency	Growth
Vehicle Control (Dimethyl Sulfoxide)	0	91	92	158	58	62
		94	98	162	58
		89	116	188	70
		92	94	174	63
Test Chemical	2.5	90#	71	135	50	56
		75	93	150	67
		99	107	150	51
	5	89	103	174	65	56
		73	103	126	58
		75	100	100	45
	10	84	100	253	100	79
		89	110	159	59
		103	111	236	76
	20	75	52	148	66	58
		89	64	144	54
		108	71	172	53
	30	71	28	239	112	96*
		84	29	216	86
		58	27	158	91
	40	62#	16	222	120	106*
		81	13	247	101
		82	14	239	98
Positive Control (Ethyl Methane Sulfonate)	250	65	49	998	510	449*
		76	56	951	415
		75	49	949	421

 

Induced S9 Trial: 1 Experiment Call: Positive

	Conc.	Cloning	Relative Total	Mutant Colonies	Mutant Frequency	Avg Mutant Frequency
	µg/mL	Efficiency	Growth
Vehicle Control (Dimethyl Sulfoxide)	0	267r	305	180	22	71
		74#	93	197	89
		98	89	182	62
		115	118	216	63
Test Chemical	0.313	84	77	203	80	81
		75	80	177	78
		80	105	199	83
	0.625	83	89	218	88	80
		94	86	222	78
		103	110	232	75
	1.25	91#	89	192	70	89
		78#	63	227	97
		94	69	279	99
	2.5	62#	41	303	164	179*
		71	28	447	210
		73	52	359	165
	5	78	26	403	171	196*
		70	17	394	189
		59	14	404	228
	10	63#	3	412.5	219	165*
		69	4	276	133
		63	8	274	145
Positive Control (3-Methylcholanthrene)	2.5	67	42	866	429	410*
		73	58	844	387
		61	61	760	413

 

Induced S9 Trial: 2 Experiment Call: Positive

	Conc.	Cloning	Relative Total	Mutant Colonies	Mutant Frequency	Avg Mutant Frequency
	µg/mL	Efficiency	Growth
Vehicle Control (Dimethyl Sulfoxide)	0	99#	127	79	27	34
		86	94	84	32
		86	107	102	39
		75	72	80	36
Test Chemical	2.5	75#	54	102	45	45
		87	69	112	43
		76	63	108	47
	5	106	35	173	54	57*
		72	35	156	72
		79#	50	103	43
	7.5	70	28	186	88	81*
		63	27	162	86
		95	32	195	68
	10	79	31	194	82	91*
		67	22	170	85
		67	21	214	107
	15	82	19	244	99	111*
		81	10	266	109
		63	14	235	124
Positive Control (3-Methylcholanthrene)	2.5	93	54	498	179	190*
		90	72	544	203
		83	77	469	188

 

Asterisks(*) indicate significant responses.

r = rejected value due to quality control criteria

# = reduced sample size because of the loss of one culture dish due to contamination

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test substance scored negative in the micronucleus test (OECD TG 474, GLP)

The test substance scored negative in the comet assay (OECD TG 489, GLP)

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:

5 Feb 2017 to 9 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): Tellurium diethyldithiocarbamate
- Batch No.: 60700109
- Purtiy: ≥ 99%
- Date of receipt: 25 Jul 2016
- Expiration date: 14 Jul 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Ambient temperature (15 - 25 °C)

OTHER SPECIFICS:
- Appearance: Yellow powder

Species:

rat

Strain:

Wistar

Remarks:

(Crl:WI(Han))

Details on species / strain selection:

- Justification for strain selection: The Wistar strain was used because it is routinely used at the test facility for this type of studies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The animals were obtained from a colony maintained under specific pathogen free (SPF) conditions by Charles River Laboratories.
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 188.5 - 234.2 g
- Assigned to test groups randomly: yes
- Housing: The animals were housed two to five animals per cage. All animals were housed in Makrolon cages with wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany) as bedding material and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. The cages and bedding were changed at least weekly. Animals treated with the positive controls were housed in filter top cages after dosing.
- Diet: Feed was provided ad libitum from the arrival of the rats until the end of the study. The animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). Each batch of diet was analyzed by the supplier for nutrients and contaminants. The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate that serves to prevent spillage. The feed in the feeders were replaced with fresh portions once weekly and filled up as needed.
- Water: Drinking water was provided ad libitum from the arrival of the rats until the end of the study. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC) was supplied by N.V. Vitens.
- Acclimation period: 11 - 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Relative humidity: 45 - 65 %. Occasionally, the relative humidity briefly exceeded 65% due to meteorological circumstances or because of wet cleaning activities.
- Air changes: 10 per hour
- Photoperiod: 12 hours light and 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: corn oil
- Concentration of test material in vehicle: 1-2.5 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Batch no.: A1600985
- Purity: 100%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
For each day of the study and for each test substance group, the appropriate amount of test substance was weighed in a glass bottle. Each dosing day, the corresponding amount of corn oil was added to obtain the final concentration of the test substance in corn oil. Before dosing, the suspension was stirred until visual homogeneity was obtained. All suspensions were continuously stirred on a magnetic stirrer during the entire daily administration period, in order to maintain the homogeneity of the test substance in the vehicle.

Duration of treatment / exposure:

Three successive days.

Frequency of treatment:

Daily

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

45 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Five. One surplus animal was treated in the same way as the five other males of these groups. These animals were finally not needed in the study and killed interim.

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C (MMC)
- Route of administration: intraperitoneally, ca. 24 h before sacrifice
- Doses / concentrations: 1.5 mg/kg bw (stock concentration 0.15 mg/mL in physiological saline; dosing volume 10 mL/kg bw)

Tissues and cell types examined:

Bone marrow / bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- The highest concentration of the test substance was intended to result in toxic effects but not death or severe suffering.
- The lowest concentration was intended to produce little or no evidence of toxicity.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Ca. 24 h interval between the first and second dose and ca. 21 h between the second and third dose. The third dose was administered ca. 3 h before scheduled sacrifice.

BONE MARROW COLLECTION AND PROCESSING
Immediately following liver perfusion (control and treatment groups) or sacrifice at ca. 24 h after intraperitoneal injection (positive control), the bone marrow cells of one of the femurs were collected into foetal calf serum and processed into glass-drawn smears according to the method described by Schmid (1976). Four bone marrow smears per animal were made, air-dried and fixed in methanol. Two fixed smears were stained with a May-Grünwald Giemsa solution. The other fixed smears were kept in reserve. If needed, these reserve slides would be stained with May-Grünwald Giemsa solution and used for microscopic examination.

MICROSCOPIC EXAMINATION OF THE BONE MARROW SMEARS
- Slides were coded by a qualified person not involved in analysing the slides to enable ‘blind’ scoring. Slides (two per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation are evenly distributed over the whole smear.
- The numbers of polychromatic erythrocyte (PE) and normochromatic erythrocyte (NE) were recorded in a total of at least 500 erythrocytes (E) per animal. If micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total of 500 E (PE + NE) have been scored, an additional number of PE was scored for the presence of micronuclei until a total of 4000 PE has been scored. In case of high cytotoxicity (<10% PE per total E) scoring of all E was continued until a total of 6000 E has been reached.

Evaluation criteria:

SCORING CRITERIA
- A polychromatic erythrocyte (PE) is an immature erythrocyte that still contains ribosomes and can be distinguished from mature, normochromatic erythrocytes by a faint blue stain.
- A normochromatic erythrocyte (NE) is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by a yellow stain.
- A micronucleus is a small, normally round, nucleus with a diameter of circa 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain.

EVALUATION CRITERIA
- The study was considered valid if the positive control group showed a statistically significant increase in the mean number of MPE/4000 PE compared to the negative control group, and if the negative control group was within the historical range.
- The test substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus if it showed a dose related positive response or a statistically significant increase of micronucleated cells in one or more dose groups when compared to the negative control group.
- The test substance was considered to be negative in the micronucleus test if it did not produce a positive response at any of the dose levels analysed.
- Biological relevance and the likelihood that the test substance or its metabolites reached the target tissues was taken into account for interpretation of the results.

Statistics:

The mean ratio PE/E and MPE/4000 PE were calculated for each group.
Two ANOVA models were applied for both PE/500E and MPE/4000PE. The first ANOVA model is used to test if the positive control differed from the negative control (t test). In a second ANOVA model, it was tested if the negative control differed from the test substance (different doses). For both models it was checked if the ANOVA assumptions were met (i.e. if variances were equal). This was the case for the second ANOVA model, but not for the first. Therefore, non-parametric testing was performed using the Mann Whitney test to if the positive control differed from the negative control. In all statistical tests a significance level of 5% was used (α = 0.05). All statistical tests were performed using GraphPad Prism®, Version 6.04, January 17, 2014. 1992-2014 GraphPad Software, Inc., CA, USA.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was no statistically significant increase in the mean numbers of MPE/4000 PE in the groups treated with the test substance when compared to the negative control group. This indicated that oral administration of the test substance, up to 45 mg/kg-bw (by gavage), did not result in damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells. Statistical analysis of the test results indicated that there was no statistically significant difference in the mean number of PE/500E when comparing the animals treated with the test substance when compared to the negative control group.

Table: Mean numbers of the PE/500 E and MPE/4000 PE observed in the bone marrow micronucleus test in rats after oral exposure to the test substance.

Treatment	PE/500 E (mean ± SD)	MPE/4000 PE (mean ± SD)
Negative control (corn oil)	259 ± 49	2.4 ± 1.8
5 mg/kg bw/day	221 ± 65	6.0 ± 3.4
15 mg/kg bw/day	197 ± 61	4.4 ± 2.3
45 mg/kg bw/day	223 ± 49	5.8 ± 2.3
Positive control (MMC)	202 ± 22*	49.6 ± 51.9**

* Statistically significantly different from negative control, P-value <0.0430 (t-test)

** Statistically significantly different from negative control, P-value 0.0397 (Mann-Whitney)

CLINICAL SIGNS AND BODY WEIGHT

No mortality was observed and no treatment-related clinical signs were observed in the animals during the study period. The few clinical signs noted (skin encrustations, and irregular respiration (a single animal of the negative control group only)) occurred randomly throughout the groups in a few animals only and are not treatment related.

Group mean body weights in all groups were considered within the normal range as expected for healthy rats of this age and strain. There were no statistically significant effects on mean body weight following treatment with the test substance.

VALIDITY OF THE RESULTS

A statistically significant decrease (P-value: 0.0430) in the mean number of PE/500 E in the positive control animals was observed, when compared to the negative control animals. This indicated that the positive control substance Mitomycin C reached the bone marrow and induced damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells of male rats. The mean numbers of MPE/4000 PE in the negative control group were within the historical range when related to 2000 PE. Furthermore, the positive control animals demonstrated a statistical significant increase in the mean number of MPE compared to the negative control animals (P-value: 0.0397). The mean number of MPE found in the positive control Mitomycin C was within historical range when related to 2000 PE. However, two out of five animals of the positive control groups showed a response in the range of the negative control. The reason for the absence of a response in these animals is unknown (probably resulted from incorrect dosing with the positive control substance, although there were no observations to confirm a dosing error). Since the other three animals clearly showed a positive response, the micronucleus test was considered valid.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Feb 2017 to 9 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): Tellurium diethyldithiocarbamate
- Batch No.: 60700109
- Purtiy: ≥ 99%
- Date of receipt: 25 Jul 2016
- Expiration date: 14 Jul 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Ambient temperature (15 - 25 °C)

OTHER SPECIFICS:
- Appearance: Yellow powder

Species:

rat

Strain:

Wistar

Remarks:

(Crl:WI(Han))

Details on species / strain selection:

- Justification for strain selection: The Wistar strain was used because it is routinely used at the test facility for this type of studies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The animals were obtained from a colony maintained under specific pathogen free (SPF) conditions by Charles River Laboratories.
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 188.5 - 234.2 g
- Assigned to test groups randomly: yes
- Housing: The animals were housed two to five animals per cage. All animals were housed in Makrolon cages with wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany) as bedding material and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. The cages and bedding were changed at least weekly. Animals treated with the positive controls were housed in filter top cages after dosing.
- Diet: Feed was provided ad libitum from the arrival of the rats until the end of the study. The animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). Each batch of diet was analyzed by the supplier for nutrients and contaminants. The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate that serves to prevent spillage. The feed in the feeders were replaced with fresh portions once weekly and filled up as needed.
- Water: Drinking water was provided ad libitum from the arrival of the rats until the end of the study. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC) was supplied by N.V. Vitens.
- Acclimation period: 11 - 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Relative humidity: 45 - 65 %. Occasionally, the relative humidity briefly exceeded 65% due to meteorological circumstances or because of wet cleaning activities.
- Air changes: 10 per hour
- Photoperiod: 12 hours light and 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: corn oil
- Concentration of test material in vehicle: 1-2.5 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Batch no.: A1600985
- Purity: 100%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
For each day of the study and for each test substance group, the appropriate amount of test substance was weighed in a glass bottle. Each dosing day, the corresponding amount of corn oil was added to obtain the final concentration of the test substance in corn oil. Before dosing, the suspension was stirred until visual homogeneity was obtained. All suspensions were continuously stirred on a magnetic stirrer during the entire daily administration period, in order to maintain the homogeneity of the test substance in the vehicle.

Duration of treatment / exposure:

Three successive days.

Frequency of treatment:

Daily.

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

45 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Five. One surplus animal was treated in the same way as the five other males of these groups. These animals were finally not needed in the study and killed interim.

Control animals:

yes, concurrent vehicle

Positive control(s):

INTESTINE
Methyl methane sulfonate (MMS)
- Route of administration: once intraperitoneally, ca. 3 hours before sacrifice.
- Doses / concentrations: 60 mg/kg-bw (stock concentration 6 mg/ml; dosing volume 10 ml/kg-bw)

LIVER
2-acetylaminofluorene (2-AAF)
- Route of administration: once orally (by gavage), ca. 12-16 hours before sacrifice.
- Doses / concentrations: 50 mg/kg-bw (stock concentration 2.5 mg/ml; dosing volume 20 ml/kg-bw)

Tissues and cell types examined:

Liver / hepatocytes and jejunum / intestinal cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- The highest concentration of the test substance was intended to result in toxic effects but not death or severe suffering.
- The lowest concentration was intended to produce little or no evidence of toxicity.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Ca. 24 h interval between the first and second dose and ca. 21 h between the second and third dose. The third dose was administered ca. 3 h before scheduled sacrifice.

ISOLATION OF HEPATOCYTES
Hepatocytes were isolated from the liver of the animals of the negative control, animals treated with the test substance and the positive control using the perfusion technique described by Williams et al. (1977) with minor modifications. Briefly, the liver of each rat were perfused in situ with a HEPES buffer (0.01 M) whilst under sodium pentobarbital anaesthesia and exsanguination from the abdominal aorta, followed by an in vitro perfusion with a HEPES-buffered (0.1 M) collagenase solution. Directly after the start of the perfusion to remove the blood from the tissue, a small part of the caudate lobe was tied off using a ligature. Subsequently, part of the lobe was removed and preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of formalin). Since a positive response was not observed, these samples were not used for histological evaluation to determine whether cytotoxicity was involved. After isolation, the dissociated cells were incubated for 5-10 minutes in a shaking water bath at 37 ºC. Thereafter, they were filtered over a nylon filter (200 μm), centrifuged and resuspended in Williams medium E. Cell counts were made and the viability of the hepatocytes were determined by trypan blue exclusion.

ISOLATION OF INTESTINAL CELLS
As soon as possible after liver perfusion or sacrifice (positive control group), the first part of the jejunum (ca. 20 cm from the stomach) of each rat was collected and kept in ice-cold Krebs-Ringer bicarbonate buffer (Sigma-Aldrich Chemie B.V., pH set at ca. 7.3) until further processing. The tissue were finely minced and homogenized in 2 mL pre-warmed (37 ºC) collagenase solution (1 mg/ml in Krebs-Ringer buffer (pH ca. 7.3), supplemented with 1.26 g/L NaHCO3, 6.3 mM CaCl2, and 1% bovine serum albumin). After a short incubation (ca. 5 min) at ambient temperature, the cell suspension was filtered twice (500 μm, followed by 70 μm) and centrifuged (500 rpm, 3 min, ca. 4 ºC). The cells were resuspended in a sufficient volume for slide preparation. The viability of the cells were determined by trypan blue exclusion. In addition, a small piece of lung was preserved in neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of formalin) for possible histopathological examination. In case of a positive response, this sample would be used for histological evaluation to determine whether cytotoxicity was involved.

PREPARATION OF SLIDES
Microscopic slides was prepared by mixing an aliquot of the cell suspension with a low-melting agarose solution (0.5 % (w/v) in Phosphate Buffered Saline). Subsequently, this mixture was loaded on a glass slide, pre-coated with normal-melting agarose (1.5 % (w/v) in PBS), and mounted with a coverslip. Three slides per animal were prepared (one slide was kept in reserve). The slides were stored on a cold plate until the agarose has solidified. Subsequently, the coverslip was removed and the slide was incubated in lysis buffer (2.5 M NaCl, 0.1 M Na2EDTA, 0.175 M NaOH, 0.01 M Tris in Milli-Q water, supplemented with 1 % Triton X-100 (w/v), pH 10) overnight at 2-10 ºC. Subsequently, slides were incubated in ice-cold electrophoresis buffer (0.3 M NaOH, 0.001 M Na2EDTA in Milli-Q water, pH >13) for 30 ± 1 min, following electrophoresis (ca. 32V and 360 mA) for 30 ± 1 min in ice-cold electrophoresis buffer, while cooled on ice. After incubation in neutralisation buffer (0.4 M Tris in Milli-Q water, pH 7.5) for at least 5 min, slides were dehydrated by incubating in ethanol at room temperature and air-dried.

SLIDE ANALYSIS AND COUNTING
Slides were coded by a qualified person not involved in analysing the slides to enable ‘blind’ scoring. Slides were stained with ethidium bromide solution (20 μg/mL in Milli-Q water) which was directly pipetted on the slide and covered with a coverslip just before analysis. A fluorescent microscope connected to a camera and Comet Assay IV software (Perceptive Instruments) was used for the analysis of the slides. Seventy-five cells (randomly selected starting from the center of the slide) per slide and two slides per animal were analysed to yield a total number of 150 cells per animal, except that for two animals (one in mid dose of test substance group and one in positive control MMS group) in total 123 and 162 intestinal cells were analysed from three slides due to very low cell density on the two comet slides that were initially analysed, respectively. For the animal in the mid dose group, only 105 cells analysed from two slides were used for calculation of the animal mean.

Ghost cells, with a small head and a diffuse and large tail, were excluded from analysis, but their presence was recorded. Microscopic slides will be retained for at least one month after finalisation of the report and then discarded.

Evaluation criteria:

EVALUATION CRITERIA
- The viability of the hepatocytes and intestinal cells of the negative control animals should be at least 50 %.
- The assay was considered valid if the group mean tail intensity of the positive control groups (for intestine and liver) showed a statistically significant increase compared to the group mean of the negative control group and if the group mean tail intensity of the negative control was comparable to the historical range.
- The test substance was considered to be positive in the in vivo comet assay if a statistically significant increase was observed at one or more dose levels compared to the group mean of the negative control group and/or if a dose related increase in the group mean tail intensity was observed. Positive results from the in vivo comet assay indicated that the test material has the potential to induce primary DNA damage in vivo in the tissue evaluated, under the conditions used in this study.
- The test substance was considered to be negative in the in vivo comet assay if no statistically significant increase was observed at any of the dose levels compared to the group mean of the negative control group. Negative results indicated that the test material did not have the potential to induce DNA damage in vivo in the tissue evaluated, under the test conditions used in this study.
- Biological relevance was taken into account for interpretation of the results. If a positive response in the comet assay was obtained, the possibility that the increase in DNA migration was not associated with genotoxicity, but with severe toxicity, was assessed. Furthermore, the likelihood that the test substance or its metabolites reached the target tissues is discussed.

Statistics:

Two ANOVA models were applied. In the first ANOVA model it was tested if the positive control differs from the negative control (unpaired t test). In a second ANOVA model, it was tested if the negative control differed from the test substance (different doses) using the Dunnett’s multiple comparisons test. For both models and for both tissues the ANOVA assumptions were met (i.e. variances were equal). In all statistical tests, a significance level of 5% was used (α = 0.05). All statistical tests were performed using GraphPad Prism®, Version 6.04, January 17, 2014. 1992-2014 GraphPad Software, Inc., CA, USA.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Tail intensity of the test substance was comparable to the negative control and did not demonstrate a statistically significant increase in tail intensity both in intestinal and liver cells at any of the concentrations tested. However, tail intensity of the test substance was just outside the historical range for intestinal cells at the lowest concentration tested. Since this increase was observed at the lowest concentration only and was not dose-related, the response was considered not biologically relevant.

Table: Mean tail intensity of intestinal and liver cells of rats after oral exposure to the test substance.

Treatment	Tail intensity intestinal cells (mean ± SD)	Tail intensity liver cells (mean ± SD)
Negative control (corn oil)	6.71 ± 2.10	3.07 ± 1.56
5 mg/kg bw/day	15.12 ± 7.37	1.90 ± 0.75
15 mg/kg bw/day	8.50 ± 5.38	2.05 ± 1.16
45 mg/kg bw/day	9.79 ± 12.09	2.06 ± 1.04
Positive control (intestine, MMS)	48.67 ± 4.95*	Not determined
Positive control liver, 2-AAF)	Not determined	15.92 ± 1.66*

*Statistically significantly different from negative control, P-value <0.0001 (t-test)

CLINICAL SIGNS AND BODY WEIGHT

No mortality was observed and no treatment-related clinical signs were observed in the animals during the study period. The few clinical signs noted (skin encrustations, and irregular respiration (a single animal of the negative control group only)) occurred randomly throughout the groups in a few animals only and are not treatment related.

Group mean body weights in all groups were considered within the normal range as expected for healthy rats of this age and strain. There were no statistically significant effects on mean body weight following treatment with the test substance.

VALIDITY OF THE RESULTS

- The percentage viability of the isolated intestinal cells was 94%, 92%, 98% and 95% for the negative control, 5 mg/kg bw, 15 mg/kg bw and 45 mg/kg bw of the test substance, respectively, whereas the percentage viability of the isolated hepatocytes was 93%, 91%, 94% and 93%, respectively. Based on the observed viability, all cell suspensions were considered suitable for the comet assay.

- The positive control substances MMS and 2-AAF demonstrated a statistically significant increase in tail intensity compared to the negative control for intestine and liver, respectively (p-value: <0.0001 for both tissues). Mean tail intensity of the negative control was within the historical range for both intestine and liver. Therefore, the comet assay was considered valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

In vitro: Reverse bacterial mutation assay (Ames test)

The mutagenic activity of the substance was evaluated in three tests similar to OECD TG 471 and according to GLP principles. In the first test (Pennwalt 1986) the dose levels (1.5-150 µg/plate) were selected based on the dose range finding experiment (5-5000 µg/plate). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA 1538, TA98, TA100) in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic with and without metabolic activation.

The NTP (NTP 1980 and 1983) performed two tests, with dose levels of 0.001 to 100 µg/plate or 0.001 to 33.0 µg/plate were selected based on cytotoxicity. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA 1538, TA98, TA100), in both tests, in the absence and presence of S9-metabolic activation.

In vitro: chromosome aberration test

The cytogenic activity of the substance was evaluated by the NTP (1983) in a test similar to OECD 473. The dose levels were 0.032, 0.1, 0.15, 0.32 µg/mL (without S9) and 0.1, 0.32, 1.0, 15.0 µg/mL (with S9). Solvent controls and positive controls were included. The substance scored positive for genotoxic activity in absence of S9 mix, and negative in presence of S9 mix.

 

In vitro: MLA

The mutagenic activity of the substance was evaluated in mammalian cells by the NTP in a study similar to OECD 490.Two trials were performed. In the first trial, the substance was tested at 1.25, 2.5, 5, 10, 20, 30, 40 µg/mL (without S9) and at 0.313, 0.625, 1.25, 2.5, 5, 10 µg/mL (with S9). In the second trial, the substance was tested at 2.5, 5, 10, 20, 30, 40 µg/mL (without S9) and at 2.5, 5, 7.5, 10, 15 µg/mL (with S9). The substance was dissolved in DMSO. Appropriate solvent and positive controls were included. The substance scored positive for mutagenic potential, both with and without S9 mix.

 

Supporting data

 

The substance was tested by the NTP (1983) for its cytogenic potential in a SCE study similar to OECD 479. The substance was tested without S9 at 0.01, 0.015, 0.02 µg/mL, and with S9 at 0.1, 0.32, 1 µg/mL. The vehicle was DMSO. Appropriate solvent and positive controls were included. The substance scored negative for cytogenic potential, both with and without S9 mix.

 

In a pre-GLP study, the mutagenic activity of the substance was evaluated similar to OECD TG 471. The dose levels were 1-1000 µg/plate. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA 1538, TA98, TA100) in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic with and without metabolic activation.

In a publication (Hedenstedt, 1979), the mutagenic activity of the substance was evaluated similar to OECD TG 471. The dose levels were 1-100 µg/plate. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in S. typhimurium tester strains TA1535, TA1537, TA 1538 in the absence and TA100 in the presence and absence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic with and without metabolic activation.

In a publication (Rannug, 1984), the mutagenic activity of the substance was evaluated similar to OECD TG 471. Dose levels were not mentioned. The substance did not induce a significant dose-related increase in the number of revertant colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA 1538, TA98, TA100) in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic with and without metabolic activation.

In vivo

In vivo: Micronucleus test and Comet Assay

The test substance was examined for its potential to cause damage to the chromosomes and/or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow in a GLP-compliant micronucleus test, conducted in accordance with OECD Guideline 474 (Triskelion B. V., 2017). In the same study, The test substance was examined for its potential to cause primary DNA damage, such as single and double strand DNA breaks, alkali labile sites and incomplete repair sides, in intestinal and liver cells in a GLP-compliant comet assay, conducted in accordance with OECD Guideline 489.

For this study, 5 male Wistar rats received 0 (control), 5, 15 and 45 mg/kg bw/day of the test substance in corn oil by oral gavage (dosing volume 5 mL/kg bw) for three successive days. There was a ca. 24 h interval between the first and second dose and ca. 21 h between the second and third dose. The third dose was administered ca. 3 h before scheduled sacrifice. All animals survived until scheduled sacrifice. No treatment-related clinical abnormalities were observed. There were no statistically significant effects on mean body weight following treatment.

Micronucleus test: 

Positive control animals for the test were intraperitoneally injected once with mitomycin C (MMC, 1.5 mg/kg bw).  Ca. 3 hours after the last administration, animals were necropsied. Bone marrow was collected, processed, microscopically examined and analysed. the group mean number of micronucleated polychromatic erythrocytes (MPE) per 4000 polychromatic erythrocytes (PE) of the negative control group (corn oil) and the positive control group (MMC) showed the expected responses. However, two out of five animals of the positive control groups showed a response in the range of the negative control. The reason for the absence of a response in these animals is unknown (probably resulted from incorrect dosing with the positive control substance, although there were no observations to confirm a dosing error). Since the other three animals clearly showed a positive response, the micronucleus test was considered valid. Results show that the mean numbers of MPE per 4000 PE and PE per 500 erythrocytes (E) of the groups treated with the test substance were comparable to the negative control (corn oil) and did not demonstrate a statistically significant increase (number MPE) or decrease (number PE) at any of the concentrations tested. Based on the results, the test substance was considered to be negative in the in vivo micronucleus test.

Comet assay:

Positive control animals for intestine and liver were orally administered (by gavage) once with methyl methanesulfonate (MMS, 60 mg/kg bw) 2-6 h prior to sacrifice or with 2-acetylaminofluorene (2-AAF, 50 mg/kg bw) 12-16 h prior to sacrifice, respectively. Ca. 3 hours after the last administration, animals were necropsied. Liver cells (hepatocytes) were isolated by liver perfusion, followed by collection of a part of the intestine (jejunum) and mincing to obtain intestinal cells and preparation of comet slides. Based on the observed viability, all cell suspensions were considered suitable for the comet assay. Tail intensity (i.e. the percentage DNA in the ‘tail’ of the comet) was used as a measure for primary DNA damage in the comet assay. The positive control substances MMS and 2-AAF demonstrated a statistical significant increase in tail intensity compared to the negative control for intestine and liver, respectively. Mean tail intensity of the negative control was within the historical range. Therefore, the comet assay was considered valid. Results show that tail intensity of the test substance was comparable to the negative control (corn oil) and did not demonstrate a statistically significant increase in tail intensity both in intestinal and liver cells at any of the concentrations tested. However, tail intensity of the test material TDEC was just outside the historical range for intestinal cells at the lowest concentration tested. Since this increase was observed at the lowest concentration only and was not dose-related, the response was considered not biologically relevant. Based on the results, the test substance was considered to be negative in the comet assay.

In vivo: SLRL test

In a publication by Valencia (1985), the test substance was tested for mutagenicity in D. melanogaster in a study similar to OECD 477. The test substance was injected in Canton-S wild-type males and potential induction of SLRLs was investigated. No significant change in SLRLs was observed. therefore, it was concluded that the test substance was not mutagenic.

Justification for classification or non-classification

Based on the negative results of the in vitro Ames test, the in vivo micronucleus test with rats and the in vivo comet assay with rats, classification of the test substance for genotoxicity is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.