long chain alkyl thio carbamide metal complex

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

EC# 457-320-2 showed no evidence of mutagenic potential in an Ames test or an in vitro gene mutation assay in mammalian cells (Mouse Lymphoma Assay), and EC# 434-650-5 - a substance structurally and chemically similar to EC# 457-320-2 - showed no evidence of genotoxic potential in an in vitro chromosome aberration study and an in vivo mouse micronucleus study. In view of the similarity of the two substances, it is concluded that further testing on EC# 457-320-2 is unnecessary and read-across to the results for EC# 434-650-5 can be justifiably used. It is therefore concluded that EC# 457-320-2 is not genotoxic.

Short description of key information:
EC# 457-320-2 showed no evidence of mutagenic potential in an Ames test or mouse lymphoma assay, and a substance structurally and chemically similar to EC# 457-320-2 showed no evidence of genotoxic potential in an in vitro chromosome aberration study and an in vivo mouse micronucleus study.

Endpoint Conclusion: No adverse effect observed (negative)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 2003 to 7 May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus (S. typimurium strains), tryptophan locus (E. coli)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial fraction derived from Aroclor 1254-induced rat liver (S-9 mix).

Test concentrations with justification for top dose:

Range-finding study: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Study 2: 0, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: hexane
- Justification for choice of solvent/vehicle: due to the insolubility of the test substance in more commonly used solvents

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 Migrated to IUCLID6: 0.5 ug/plate (TA 1535 and TA100)

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 Migrated to IUCLID6: 50 ug/plate (TA1537)

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 Migrated to IUCLID6: 1 ug/plate (TA98)

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; 0.05 ug/plate (WP2 uvrA, pKM101)

Remarks:

without S9

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; 2 ug/plate (TA1535), 10 ug/plate (WP2 uvrA pKM101)

Remarks:

with S9

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 Migrated to IUCLID6: 5 ug/plate (TA1537, TA98, TA100)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: none
- Exposure duration: 72 hr


NUMBER OF REPLICATIONS: 3 plates prepared per concentration, two independent studies were carried out

NUMBER OF CELLS EVALUATED: 10(8)/plate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (background lawn)

Evaluation criteria:

Considered positive if there was a reproducible increase in revertants of an least twice (three times for TA1535 and TA1537) the vehicle controls.

Statistics:

No statistical analysis was performed where the test gave a clear positive or negative result

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Remarks:

There is historical control data on the vehicle, therefore untreated control not necessary in accordance with the OECD TG 471.

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Remarks:

There is historical control data on the vehicle, therefore untreated control not necessary in accordance with the OECD TG 471.

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Remarks:

There is historical control data on the vehicle, therefore untreated control not necessary in accordance with the OECD TG 471.

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Remarks:

There is historical control data on the vehicle, therefore untreated control not necessary in accordance with the OECD TG 471.

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Remarks:

There is historical control data on the vehicle, therefore untreated control not necessary in accordance with the OECD TG 471.

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: the first study was a range-finding study

COMPARISON WITH HISTORICAL CONTROL DATA: historical control data was included in the report for the bacterial strains used, the combined data for solvent controls, and for the positive controls

Remarks on result:

other: No mutagenic potential

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

Conclusions:

Interpretation of results: negative

In a GLP study conducted according to OECD guideline 471, test material MRD-02-375 (EC# 457-320-2) showed no mutagenic potential in an assay for reverse mutation in bacterial strains, at up to 5 mg/plate with and without S9.

Executive summary:

In a GLP study conducted according to OECD guideline 471, the mutagenic potential of test material MRD-02 -375 (EC#457-320-2) was assessed in a bacterial mutation assay (Ames test).

Up to 5000 micrograms/plate of the test substance (dissolved in hexane) was incubated with S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2 uvrA pKM101 in a plate incorporation assay, both with an without a rat liver activation fraction (S9). After 72 hr incubation the revertant colonies were counted using an automated colony counter. Two independent assays were carried out with triplicate plates being prepared for each concentration.

No increases in revertant colonies were observed with the test material compared to the vehicle controls. No cytotoxicity was observed as shown by the presence of a confluent background lawn. The positive control substances showed the expected increases in mutant frequency, indicating the effective performance of the assay.

In conclusion, EC# 457 -320 -2 showed no mutagenic potential in an assay for reverse mutation in bacterial strains, at up to 5 mg/plate with and without S9.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 February to 12 April 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial fraction derived from the liver of Aroclor 1254-induced rats (S-9)

Test concentrations with justification for top dose:

Study 1 (without S9): 0, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml
(with S9): 0, 50, 100, 150, 200, 250, 300, 400, 600 and 800 µg/ml

Study 2 (without S9): 0, 125, 250, 750, 1000, 2000, 3000 and 4000 µg/ml
(with S9): 0, 50, 100, 150, 200, 250, 300, 400, 600 and 800 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: insoluble in water and acetone, but soluble in culture medium to about 10 mg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 Migrated to IUCLID6: 0.1 µg/ml

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Migrated to IUCLID6: 6 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: (study 1): 3 hr both with and without S9;
(study 2): 3 hr with S9, 21 hr without S9
- Fixation time (start of exposure up to fixation or harvest of cells): 21 hr


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 100 per culture (200 per dose level)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

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

Evaluation criteria:

The assay was considered positive if statistically significant increases (P < 0.01) in the frequency of aberrations were observed at one or more concentrations and the increases were reproducible between the replicate cultures

Statistics:

Fisher exact test

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: study 1 was a range-finding study

COMPARISON WITH HISTORICAL CONTROL DATA: historical data on spontaneous levels and positive controls for January 1997 to December 1998 given in report

ADDITIONAL INFORMATION ON CYTOTOXICITY: see Table 1

Based on the mitotic indices the dose levels selected for metaphase analysis were:
Study 1: 1250, 2500 and 5000 µg/ml (without S9)
78.13, 156.25 and 312.5 µg/ml (with S9)
Study 2: 2000, 3000 and 4000 µg/ml (without S9)
400, 600 and 800 µg/ml (with S9)

There was no increase in polyploidy in treated cultures compared to the vehicle control.

Responses obtained from the negative and positive controls demonstrated that the system was capable of detecting chemicals that caused chromosomal damage.

Remarks on result:

other: No mutagenic potential

Table 1. Summary of results

Exposure period (hr)	S9 mix	Concentration of test substance (µg/ml)	Cells with CAs excluding gaps (%)	Cells with CAs including gaps (%)	Mitotic index (%)
Study 1
3	-	0	0	1.0	100
		1250	0.5	1.5	94
		2500	0	3.0	78
		5000	0.5	1.0	37
		0.1 mitomycin C	13.5***	20.0***	-
3	+	0	0	1.0	100
		78.13	0.5	2.0	93
		156.25	1.0	2.0	114
		312.5	0	0.5	29
		6 cyclophosphamide	10.5***	19.5***	-
Study 2
21	-	0	0.5	0.5	100
		2000	0.5	1.0	92
		3000	0.5	1.5	85
		4000	1.0	2.0	58
		0.1 mitomycin C	13.5***	21.0***	-
3	+	0	0.5	0.5	100
		400	0.5	1.0	96
		600	0.5	1.5	81
		800	1.0	2.0	59
		6 cyclophosphamide	10.5***	17.0***	-

*** P <0.001

 

 

 

Conclusions:

Interpretation of results: negative

In a GLP study conducted according to OECD guideline 473, EC# 434-650-5 - a substance structurally and chemically similar to EC# 457-3320-2 - showed no ability to induce chromosome aberrations or polyploidy in primary cultures of human lymphocytes, when tested at up to 800 or 5000 µg/ml with or without S9, respectively.

Executive summary:

In a GLP study conducted according to OECD guideline 473, the ability of EC# 434-650-5 (tris(dicocodithiocarbamato) tris(µ-sulfido) (µ3-thio)-triangulotrimolybdenum (IV) dicocothiocarbamate), a substance structurally and chemically similar to EC# 457-320-2, to cause chromosome aberrations was assessed in cultures of human lymphocytes.

Lymphocytes, obtained from healthy male volunteers, were grown in culture medium for 48 hr before the addition of the test substance, in the presence and absence of a rat liver metabolic activation fraction (S9). Two independent studies were carried out. In the first, up to 5000 or 800 µg/ml were incubated without or with S9, respectively, for 3 hr, before removal of the test substance and a further 18 hr incubation. In the second study up to 4000 or 800 µg/ml were incubated without or with S9, respectively, but in this case the cultures without S9 were exposed to the test substance for the entire 21 hr incubation period. Cell division was stopped in metaphase 2 hr before the end of the incubation period, after which the cells were fixed, stained with Giemsa and slides prepared. The proportion of mitoses (mitotic index) per 1000 cells were recorded and used to select the dose levels for scoring of chromosome aberrations. One hundred metaphases were scored for each duplicate culture.

No statistically significant increases in the number of chromosome aberrations or induction of polyploidy were observed with the test substance compared to the vehicle controls. Cytotoxicity was apparent as shown by the decrease in the mitotic indices of the treated cells at the higher dose levels, both with and without S9. The positive controls gave the expected response demonstrating the sensitivity of the test system.

In conclusion, EC# 434-650-5 showed no ability to induce chromosome aberrations or polyploidy in primary cultures of human lymphocytes, when tested at up to 800 or 5000 µg/ml with or without S9, respectively.

In view of the structural and chemical similarities, it is considered that the results of this study can be used for read-across to EC# 457-320-2.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: No mutagenic potential

Conclusions:

Interpretation of results: negative

In a GLP study conducted according to OECD guideline 473, EC# 434-650-5 - a substance structurally and chemically similar to EC# 457-3320-2 - showed no ability to induce chromosome aberrations or polyploidy in primary cultures of human lymphocytes, when tested at up to 800 or 5000 µg/ml with or without S9, respectively.

Executive summary:

In a GLP study conducted according to OECD guideline 473, the ability of EC# 434-650-5 (tris(dicocodithiocarbamato) tris(µ-sulfido) (µ3-thio)-triangulotrimolybdenum (IV) dicocothiocarbamate), a substance structurally and chemically similar to EC# 457-320-2, to cause chromosome aberrations was assessed in cultures of human lymphocytes.

Lymphocytes, obtained from healthy male volunteers, were grown in culture medium for 48 hr before the addition of the test substance, in the presence and absence of a rat liver metabolic activation fraction (S9). Two independent studies were carried out. In the first, up to 5000 or 800 µg/ml were incubated without or with S9, respectively, for 3 hr, before removal of the test substance and a further 18 hr incubation. In the second study up to 4000 or 800 µg/ml were incubated without or with S9, respectively, but in this case the cultures without S9 were exposed to the test substance for the entire 21 hr incubation period. Cell division was stopped in metaphase 2 hr before the end of the incubation period, after which the cells were fixed, stained with Giemsa and slides prepared. The proportion of mitoses (mitotic index) per 1000 cells were recorded and used to select the dose levels for scoring of chromosome aberrations. One hundred metaphases were scored for each duplicate culture.

No statistically significant increases in the number of chromosome aberrations or induction of polyploidy were observed with the test substance compared to the vehicle controls. Cytotoxicity was apparent as shown by the decrease in the mitotic indices of the treated cells at the higher dose levels, both with and without S9. The positive controls gave the expected response demonstrating the sensitivity of the test system.

In conclusion, EC# 434-650-5 showed no ability to induce chromosome aberrations or polyploidy in primary cultures of human lymphocytes, when tested at up to 800 or 5000 µg/ml with or without S9, respectively.

In view of the structural and chemical similarities, it is considered that the results of this study can be used for read-across to EC# 457-320-2.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2020 - February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

45% test item in 55% base oil

Target gene:

Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Obtained from the American Type Culture Collection (repository number CRL-9518), Manassas, VA.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

E00350-359 was evaluated at concentrations of 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL with 4-hour treatment with and without S9 and 3.55, 7.10, 14.2, 28.4, 56.8, 114, 227, 455 and 909 µg/mL with 24-hour treatment without S9. Visible precipitate was observed at 31.3 ug/mL and above at 4-hour treatment with and without S9, and at 14.2 ug/mL and above for 24-hour treatment without S9. Relative suspension growth (RSG) was 22, 53 and 39% at concentrations of 500 µg/mL (4-hour treatment with S9), 125 µg/mL (4-hour treatment without S9) and 114 µg/mL (24-hour treatment without S9), respectively. RSG was or approximated 0% at all higher concentrations using all treatment conditions. Although RSG at 909 µg/mL was at 47% for 24-hour treatment without S9, due to solubility limitations and precipitation profile, three lowest precipitating concentrations were chosen for the mutagenicity assay.

Therefore, based on the results from the preliminary toxicity assay, the following concentrations were selected for the definitive mutagenicity assay:
Non-activated 4-hour treatment concentrations: 1.95, 3.91, 7.81, 15.6, 31.3, 62.5 and 125 ug/mL
Non-activated 24-hour treatment concentrations: 1.78, 3.55, 7.10, 14.2, 28.4, 56.8 and 114 ug/mL
S9-activated 4-hour treatment concentrations: 1.95, 3.91, 7.81, 15.6, 31.3, 62.5 and 125 ug/mL

Vehicle / solvent:

THF was the vehicle of choice based on the solubility of the test article and compatibility with the target cells. The test article formed a clear solution in THF at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance. The formulation prepared in the solubility test also was adjusted using a correction factor of 2.22.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

THF

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

Seven concentrations were tested using duplicate cultures at appropriate dose intervals based on the precipitation profile of the test article. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. Precipitation was determined with the unaided eye at the beginning and end of treatment. When plating untreated controls, the addition of test article, vehicle and positive control were omitted.

The preparation and addition of the test article dose formulations was carried out under filtered lighting during the exposure period. Treatment was carried out by combining 50 µL of test article dose formulation (for 4 hour treatment conditions) and 20 µL of test article dose formulation (for 24 hour treatment condition) or 100 µL of positive control dose formulation and F0P medium or S9 mix (as appropriate) with 6 x 106 L5178Y/TK+/- cells in a total volume of 10 mL. All pH adjustments were performed prior to adding S9 or target cells to the treatment medium. Each S9-activated 10-mL culture contained 4 mL S9 mix (final S9 concentration of 1.0%). Cultures were capped tightly and incubated with mechanical mixing at 37 ± 1°C for 4 or 24 hours.

For the definitive assay only, at the end of the exposure period, the cells were washed with culture medium and collected by centrifugation. The cells were resuspended in 20 mL F10P on Day 1 and in 10 mL F10P on Day 2, and incubated at 37 ± 1°C for two days following treatment.

Cells from selected dose levels were cultured in triplicate with 2-4 µg TFT/mL at a density of 1 x 106 cells/100 mm plate in cloning medium containing 0.22 to 0.24% agar. For estimation of cloning efficiency at the time of selection of those same cultures, 200 cells/100 mm plate were cultured in triplicate in cloning medium without TFT (viable cell (VC) plate). Cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 11 to 12 days.
The total number of colonies per culture was determined for the VC plates and the total relative growth calculated. The total number of colonies per TFT plate was then determined for those cultures with =10% total growth (including at least one concentration between 10 and 20% total growth, if possible). Colonies were counted and the diameter of the TFT colonies from the positive control and vehicle control cultures were determined over a range from 0.2 to 1.1 mm.

Evaluation criteria:

The average spontaneous mutant frequency of the vehicle control cultures must be within 35 to 140 TFT-resistant mutants/106 surviving cells. Low spontaneous mutant frequencies, i.e., 20 to 34 mutants/106 surviving cells, were considered acceptable if small colony recovery was demonstrated (Mitchell et al., 1997). The average cloning efficiency of the vehicle controls must be between 65 and 120% and the total suspension growth between 8 to 32 for the 4-hour exposure and 32 to 180 for the 24-hour exposure (Moore et al., 2002, 2006, and 2007).
The mutant frequency for at least one dose of each positive control must meet the criteria for a positive response. The mutant frequency for at least one dose of one of the positive controls must induce an increase in small colony mutants according to the following criteria: Induced Mutant Frequency (IMF) positive control =300 x 10-6 mutants with 40% small colonies or small colony IMF for positive control =150 x 10-6 (Moore et al., 2002; 2006).
Cultures treated with a minimum of four concentrations of test article must be evaluated and their mutant frequencies reported. Results may be accepted, with justification, when only three concentrations of test article are evaluated and otherwise meet the other criteria for a valid test. The highest test article concentration must produce 80 to 90% toxicity unless limited by solubility or the maximum required concentration. In the case of a test article with a steep toxicity curve (no concentrations with 10 to 20% survival), the results may be considered acceptable if a concentration spacing of =2-fold is used and the highest concentration tested showed <20% survival or total kill (Sofuni et al., 1997).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test article did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).
Cultures treated at concentrations of 3.91, 7.81, 15.6, 31.3 and 62.5 µg/mL (4-hour treatment with S9), 3.91, 7.81, 15.6, 31.3 and 62.5 µg/mL (4-hour treatment without S9) and 3.55, 7.10, 14.2, 28.4 and 56.8 µg/mL (one replicate plate for 56.8 µg/mL as the other plate was lost due to dilution error) (24-hour treatment without S9) exhibited 107% to 120%, 76% to 102% and 51% to 97% RSG, respectively, and were cloned. Other concentrations were not evaluated as sufficient number of concentrations were available for evaluation. Relative total growth of the cloned cultures ranged from 96% to 122% (4-hour treatment with S9), 80% to 116% (4-hour treatment without S9) and 56% to 103% (24-hour treatment without S9). At least four concentrations were available for evaluation. No dose dependent increases in induced mutant frequency =90 mutants/106 clonable cells were observed under any treatment condition. No statistical trend was observed with Cochran Armitage test.
Trifluorothymidine-resistant colonies for the positive and vehicle control cultures, were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.
All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

Remarks on result:

other: No mutagenic potential via gene mutations in mammalian cells

Conclusions:

Under the conditions of the assay described in this report, E00350-359 was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

Executive summary:

The test article, E00350-359, was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Tetrahydrofuran (THF) was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL with 4-hour treatment with and without S9 and 3.55, 7.10, 14.2, 28.4, 56.8, 114, 227, 455 and 909 µg/mL with 24-hour treatment without S9. The maximum concentration evaluated approximated the limit dose for this assay for 4-hour treatment condition, and the maximum concentration evaluated was based on solubility limitations of the test article in the vehicle for 24-hour treatment condition. Visible precipitate was observed at concentrations =31.3 µg/mL (4-hour treatment conditions), =14.2 µg/mL (24-hour treatment conditions) at the beginning of treatment and at concentrations =31.3 µg/mL (4-hour treatment condition), =28.4 µg/mL (24-hour treatment condition) by the end of treatment. Relative suspension growth (RSG) was 22, 53 and 39% at concentrations of 500 µg/mL (4-hour treatment with S9), 125 µg/mL (4-hour treatment without S9) and 114 µg/mL (24-hour treatment without S9), respectively. RSG was or approximated 0% at all higher concentrations using all treatment conditions. Although RSG at 909 µg/mL was at 47% for 24-hour treatment without S9, due to solubility limitations and precipitation profile, three lowest precipitating concentrations were chosen for the mutagenicity assay. Based upon these results, the concentrations chosen for the definitive mutagenicity assays were 1.95, 3.91, 7.81, 15.6, 31.3, 62.5 and 125 µg/mL (4-hour treatment with S9), 1.95, 3.91, 7.81, 15.6, 31.3, 62.5 and 125 µg/mL (4-hour treatment without S9) and 1.78, 3.55, 7.10, 14.2, 28.4, 56.8 and 114 µg/mL (24-hour treatment without S9).

Initial definitive mutagenicity assay was not continued as sufficient cell growth was not available. The entire assay was repeated.

In the retest of definitive mutagenicity assay, visible precipitate was observed at concentrations =62.5 µg/mL (4-hour treatment conditions), =56.8 µg/mL (24-hour treatment condition) at the beginning of treatment and by the end of treatment. Cultures treated at concentrations of 3.91, 7.81, 15.6, 31.3 and 62.5 µg/mL (4-hour treatment with S9), 3.91, 7.81, 15.6, 31.3 and 62.5 µg/mL (4-hour treatment without S9) and 3.55, 7.10, 14.2, 28.4 and 56.8 µg/mL (one replicate plate at 56.8 µg/mL as the other plate was lost due to dilution error) (24-hour treatment without S9) exhibited 107% to 120%, 76% to 102% and 51% to 97% RSG, respectively, and were cloned. Other concentrations were not evaluated as sufficient number of concentrations were available for evaluation. Relative total growth of the cloned cultures ranged from 96% to 122% during the 4-hour treatment with S9, 80% to 116% during the 4-hour treatment without S9 and 56% to 103% during the 24-hour treatment without S9. At least four concentrations were available for evaluation. No dose dependent increases in induced mutant frequency =90 mutants/106 clonable cells were observed under any treatment condition. No statistical trend was observed with Cochran Armitage test.

These results indicate E00350-359 was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

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:

26 May to 2 June 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

only one sampling time, a second sampling time is recommended where two or more daily doses are given

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: about 9 weeks
- Weight at study initiation: males: 30.1-35.3 g; females: 22.6-28.3 g
- Assigned to test groups randomly: yes, using computer-generated body weight sorting programme
- Fasting period before study: no data
- Housing: singly in suspended stainless steel cages
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Peanut oil
- Vehicle(s)/solvent(s) used: peanut oil
- Justification for choice of solvent/vehicle: test substance soluble in peanut oil
- Concentration of test material in vehicle: 50, 100 and 200 mg/ml
- Amount of vehicle (if gavage or dermal): 10.0 ml/kg bw
- Lot/batch no. (if required): 116H1037
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: dosing solutions prepared once and used for all 3 days of dosing

Duration of treatment / exposure:

3 days

Frequency of treatment:

daily for 3 days

Post exposure period:

18-24 hr after the last dose

Remarks:

Doses / Concentrations:
0, 472, 939 or 1640-1930 mg/kg bw/day
Basis:
actual ingested
analytical dose

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control with peanut oil only

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control substance: cyclophosphamide
- Justification for choice of positive control(s): included in list of recommended substances
- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg bw/day

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on range-finding study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): treated daily for 3 days with 0, 500, 1000 or 2000 mg/kg bw/day. Samples taken 18-24 hr after the last dose.

DETAILS OF SLIDE PREPARATION: bone marrow aspirated from the femurs, washed, centrifuged and the erythrocytes resuspended in any remaining supernatant. Bone marrow smears were prepared (2/animal) and the slides stained with acridine orange.

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei. The percentage of PCEs were determined by counting 1000 erythrocytes per animal.

Evaluation criteria:

The test substance was considered positive if the mean incidence of micronucleated PCEs showed a dose-related increase, including at least one dose that was statistically different from the mean incidence in the vehicle control groups and outside the normal mean range of the vehicle controls (ie. greater than 4), or at least two dose levels are statistically different from the normal range for the vehicle control (again, greater than 4).

Statistics:

Standard one-way analysis of variance (ANOVA) and, if this was significant, Duncan's multiple range test was used to compare vehicle and treated groups. A standard regression analysis was performed to test for dose-response.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity: Evidence of cytotoxicity was observed at 1000 mg/kg bw/day (males and females) and 2000 mg/kg bw/day (males only) as indicated by a significant decrease in PCE/NCE ratio compared with controls.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 0, 500, 1000 or 2000 mg/kg bw/day
- Solubility: yes
- Clinical signs of toxicity in test animals: none observed, and body weights did not change during the study.
- Evidence of cytotoxicity in tissue analyzed: none observed
- Rationale for exposure: no data
- Harvest times: 18-24 hr after the last dose


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction in the treated groups
- Ratio of PCE/NCE (for Micronucleus assay): see Table 1 (below)
- Statistical evaluation: not applicable, no induction of micronuclei

Table 1. Mean number of micronuclei and PCE:NCE ratios per treatment

	Males (mean of 5 animals)		Females (mean of 5 animals)
	%PCE (ratio PCE:NCE)	Micronuclei/100PCEs	%PCE(ratio PCE:NCE)	Micronuclei/100PCEs
0 mg/kg bw/day (vehicle control)	54.46 ± 3.34 (1.18)	0.8 ± 0.8	53.14 ± 1.63 (1.13)	1.2 ± 0.7
500 mg/kg bw/day	49.68 ± 4.28 (0.99)	1.2 ± 0.4	51.32 ± 4.41 (1.05)	0.4 ± 0.2
1000 mg/kg bw/day	47.88 ± 5.64** (0.92)	0.4 ± 0.2	45.64 ± 5.56* (0.84)	0.7 ± 0.8
2000 mg/kg bw/day	44.54 ± 2.87** (0.80)	1.3 ± 0.6	47.40 ± 5.11 (0.90)	1.4 ± 0.7
20 mg/kg bw/day cyclophosphamide	36.78 ± 3.90** (0.58)	9.9 ± 2.2**	36.66 ± 6.65** (0.60)	11.3 ± 1.6**

* p < 0.05; ** p < 0.01

Conclusions:

Interpretation of results: negative
In a GLP study conducted according to OECD guideline 474, test substance MRD-98-158 (EC#434-650-5) showed no genotoxic potential in an in vivo bone marrow micronucleus assay when given orally to male and female mice at doses of up to 2000 mg/kg bw/day for three consecutive days.

Executive summary:

In a GLP study conducted according to OECD guideline 474, the genotoxic potential of test substance MRD-98-158 (EC# 434-650-5) was assessed in an in vivo bone marrow micronucleus assay in mice.

Groups of five CD-1 mice of each sex were dosed via gavage with 0, 500, 1000 or 2000 mg/kg bw/day for three consecutive days with the test substance in peanut oil. Between 18-24 hr after the final dose, bone marrow erythrocytes were recovered from the femurs of the animals and used to prepare two slides per animal for examination. After staining with acridine orange, 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. To determine cytotoxicity the number of polychromatic erythrocytes in the total erythrocyte population were counted.

No increase in micronucleated cells was evident in the treated groups compared to the vehicle controls. Cytotoxicity, indicated by a reduction in the ratio of polychromatic erythrocytes to normochromatic erythrocytes, was detected at the mid- and high-dose levels in males and the mid-dose in females. The positive controls gave the expected increase in micronuclei demonstrating the effective performance of the assay.

In conclusion, EC# 434-650-5 showed no genotoxic potential in an in vivo bone marrow micronucleus assay when given orally to male and female mice at doses of up to 2000 mg/kg bw/day for three consecutive days.

In view of the structural and chemical similarities, it is considered that the results of this study can be used for read-across to EC# 457-320-2.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: No genetic toxicity

Conclusions:

Interpretation of results: negative
In a GLP study conducted according to OECD guideline 474, test substance MRD-98-158 (EC#434-650-5) showed no genotoxic potential in an in vivo bone marrow micronucleus assay when given orally to male and female mice at doses of up to 2000 mg/kg bw/day for three consecutive days.

Executive summary:

In a GLP study conducted according to OECD guideline 474, the genotoxic potential of test substance MRD-98-158 (EC# 434-650-5) was assessed in an in vivo bone marrow micronucleus assay in mice.

Groups of five CD-1 mice of each sex were dosed via gavage with 0, 500, 1000 or 2000 mg/kg bw/day for three consecutive days with the test substance in peanut oil. Between 18-24 hr after the final dose, bone marrow erythrocytes were recovered from the femurs of the animals and used to prepare two slides per animal for examination. After staining with acridine orange, 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. To determine cytotoxicity the number of polychromatic erythrocytes in the total erythrocyte population were counted.

No increase in micronucleated cells was evident in the treated groups compared to the vehicle controls. Cytotoxicity, indicated by a reduction in the ratio of polychromatic erythrocytes to normochromatic erythrocytes, was detected at the mid- and high-dose levels in males and the mid-dose in females. The positive controls gave the expected increase in micronuclei demonstrating the effective performance of the assay.

In conclusion, EC# 434-650-5 showed no genotoxic potential in an in vivo bone marrow micronucleus assay when given orally to male and female mice at doses of up to 2000 mg/kg bw/day for three consecutive days.

In view of the structural and chemical similarities, it is considered that the results of this study can be used for read-across to EC# 457-320-2.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

EC# 457-320-2 showed no evidence of mutagenic potential in an Ames test or mouse lymphoma assay, and EC# 434-650-5 - a substance structurally and chemically similar to EC# 457-320-2 - showed no evidence of genotoxic potential in an in vitro chromosome aberration study and an in vivo mouse micronucleus study. In view of the similarity of the two substances, it is concluded that further testing on EC# 457-320-2 is unnecessary and read-across to the results for EC# 434-650-5 can be justifiably used. It is therefore concluded that EC# 457-320-2 is not genotoxic. Consequently, in accordance with the CLP and GHS Regulations no classification is proposed for genetic toxicity.