3-iodo-2-propynyl butylcarbamate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is not considered mutagenic in two bacterial reverse mutation assays applying five S. typhimurium strains, respectively.

Based on results of an unscheduled DNA synthesis assay with primary rat hepatocyte cell cultures it is concluded that the test item does not induce unscheduled DNA synthesis.

Furthermore, the test item is not considered as mutagenic in an mammalian cell mutagenicity assay (HPRT) in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1984-05-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

please refer to "principles of method if other than guideline"

Principles of method if other than guideline:

Five Salmonella strains were tested. However, a strain in order to detect cross-linking mutagens was not applied (e .g. S. typhimurium TA102 or E. coil WP2). Only plate incorporation test was performed, no preincubation test.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

mammalian S9 Mix

Test concentrations with justification for top dose:

500; 166.7; 55.6; 18.5; 6.2 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Remarks:

bacteria only and S9 fraction

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: Methylnitronitrosoguanidine; 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- relative total growth

Rationale for test conditions:

preliminary cytotoxicity test

Evaluation criteria:

Before an agent is reported to be either active or inactive in the Salmonella/microsomal assay, the following criteria must be met:

1. Demonstration of toxicity of the chemical for the bacterial strain(s), unless this is not possible due to a limited solubility of the test compound.

2. The spontaneous revertants for each strain must be within acceptable limits. The range of observed values are given below. The figures are from tests conducted in our laboratory compiled to the present.

3a. Without metabolic activation:
TA1535: Range: 10.0- 50.0; Mean: 22.7; Standard Deviation (SD): 7.5
TA1537: Range: 5.0- 19.0; Mean: 8.2; SD: 3.2
TA1538: Range: 10.0- 30.0; Mean: 13.5; SD: 4.4;
TA100: Range: 100.0-250; Mean: 130.6; SD: 39.4
TA98: Range: 15.0- 45.0; Mean: 27.6; SD: 14.6

3b. With metabolic activation:
TA1535: Range: 4.0- 26.0; Mean: 15.8; Standard Deviation (SD): 5.4
TA1537: Range: 4.0- 20.0; Mean: 9.3; SD: 3.6
TA1538: Range: 13.0- 39.0; Mean: 27.9; SD: 7.0
TA100: Range: 100.0-250; Mean: 138.9; SD: 37.6
TA98: Range: 15.0- 50.0; Mean: 38.5; SD: 10.6

4. The solvent controls must have approximately the same number of colonies as spontaneous reversion controls.

5. Positive mutagens must give at least 2x the number of colonies as the controls for spontaneous reversion. Any test with a strain which does not meet these criteria must be repeated on a separate day.

All criteria noted above must be met before results with an unknown agent can be evaluated. A chemical that exhibits a positive dose response over three concentrations with the smallest of these increases equal to twice the solvent control is considered to be mutagenic in the S. typhimurium assay.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 55.6 µg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 166.7 µg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 166.7 µg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 166.7 µg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 55.6 µg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1 results of bacterial reverse mutation assay applying plate incorporation test with Salmonella typhimurium strains.

Dose (µg/plate)	Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium
	TA 1535	TA 1537	TA 1538	TA 100	TA98
	Results without S9
DMSO	17.0 ± 1.0	14.0 ± 2.0	18.0 ± 1.7	175.3 ± 5.1	19.0 ± 4.0
Bacteria only	21.0 ± 2.6	10.7 ± 0.6	14.0 ± 6.0	222.3 ± 20.6	27.0 ± 6.1
500	T	T	T	T	T
166.7	T	2.7 ± 2.5	T	T	T
55.6	4.7 ± 2.1	6.3 ± 2.3	9.3 ± 1.5	107.7 ± 5.5	11.0 ± 4.6
18.5	16.0 ± 5.2	12.0 ± 3.0	15.7 ± 0.6	169.0 ± 7.5	20.7 ± 4.7
6.2	17.7 ± 2.1	9.0 ± 2.0	16.3 ± 4.7	191.7 ± 15.0	26.7 ± 2.1
MNNG (5)	1389.0 ± 160.7	NT	NT	2328.7 ± 87.8	NT
9- AA (75)	NT	1104.0 ± 145.7	NT	NT	NT
2-NF (50)	NT	NT	1530.7 ± 13.3	NT	1958.0 ± 45.7
	Results with S9
DMSO	13.7 ± 1.5	11.3 ± 0.6	26.3 ± 4.2	206.3 ± 24.0	32.0 ± 7.0
Bacteria only	21.0 ± 2.6	10.7 ± 0.6	14.0 ± 6.0	222.3 ± 20.6	27.0 ± 6.1
500	T	T	T	T	T
166.7	T	8.0 ± 3.0	T	51.0 ± 11.8	T
55.6	15.3 ± 2.1	9.7 ± 2.3	25.7 ± 1.2	140.3 ± 30.2	25.0 ± 6.6
18.5	14.3 ± 0.6	11.3 ± 2.5	25.3 ± 0.6	170.0 ± 11.8	32.7 ± 8.5
6.2	11.7 ± 2.5	14.3 ± 1.5	32.7 ± 2.5	179.3 ± 31.9	37.0 ± 12.1
2- AA (5)	452.3 ± 35.6	186.7 ± 9.6	2109.7 ± 145.7	2284.3 ± 20.1	2141.0 ± 83.6
S9 fraction only	14.0 ± 5.3	10.7 ± 1.5	28.0 ± 10.4	210.5 ± 21.9	42.7 ± 7.4

Conclusions:

The validity criteria were satisfied by the test conditions used with the test material. Under these conditions, the test material is not mutagenic in this test system.

Executive summary:

The purpose of this study was to examine the test item for mutagenic activity in the Salmonella typhimuriurn/Mammalian Microsome mutagenicity test. Five S. typhimurium strains, TA1535, TA 1537, TA1538, TA100 and TA98 were tested, both with and without mammalian S9 mix for metabolic acitvation. Concentrations of 500, 166.7, 55.6, 18.5 and 6.2 µg/plate were applied. Highest concentrations appeared to be cytotoxic in all strains tested. Based on the results of this study no mutagenic potential of the test item was observed.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989-06-20

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1989

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

please refer to "principles of method if other than guideline"

Principles of method if other than guideline:

Five Salmonella strains were tested. However, a strain in order to detect cross-linking mutagens was not applied (e .g. S. typhimurium TA102 or E. coil WP2). Only plate incorporation test was performed, no preincubation test.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: P-2848-8603-P100

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

arocolor induced rat liver S9 mix

Test concentrations with justification for top dose:

Toxicity test:
33, 100, 333, 1000, 3333 and 10000 µg per plate

Mutation test I:
1, 3, 10, 33, 100 and 333 µg per plate

Mutation test II:
With S9: 3, 10, 33, 100, 333 and 1000 µg per plate
Without S9: 1, 3, 10, 33, 100 and 333 µg per plate; except TA 1535 which ranged between 3 and 1000 µg per plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: recommended by the guideline

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: approx. 2 x 10^9 cells/mL

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficency

Rationale for test conditions:

according to guidelines

Evaluation criteria:

A test was considered acceptable if for each strain:
- the bacteria demonstrated their typical responses to crystal violet, ampicillin and u.v. light
- at least two of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and TA 1538, 5-35
- on at least two of the positive control plates there were x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, x 1.5 the mean vehicle control mutant numbers per plate
- if the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required on at least 2 of the positive control plates
- no toxicity or contamination was observed in at least 4 dose levels
- in cases where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number.

Where these criteria were met, a significant mutagenic response was recorded if there was:
- for S. typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98, at least a doubling of the mean concurrent vehicle control values at some concentration of the test substances and, for S. typhimurium strain TA 100, a 1.5-fold increase over the control value. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
- a dose related response, although at high dose levels this relationship could be inverted because of, for example, (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolizing enzymes where mutagens require metabolic activation by the liver.
- a reproducible effect in independent tests.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the concentration range of 333 to 1000 µg per plate without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the concentration range of 333 to 1000 µg per plate without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the concentration range of 333 to 1000 µg per plate without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the concentration range of 333 to 1000 µg per plate without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the concentration range of 333 to 1000 µg per plate 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: yes at concentration of 1000 µg per plate onwards

RANGE-FINDING/SCREENING STUDIES: yes

Remarks on result:

other: Test I and Test II

Table 1 Results of Test I

Dose (µg/plate)	Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium
	TA 1535	TA 1537	TA 1538	TA 98	TA 100
	Results without S9
DMSO	8 ± 4	11 ± 2	18 ± 5	30 ± 6	112 ± 21
1	12 ± 2	8 ± 4	15 ± 6	33 ± 3	102 ± 14
3	7 ± 4	6 ± 3	16 ± 3	33 ± 6	97 ± 4
10	8 ± 4	6 ± 2	13 ± 2	37 ± 2	88 ± 13
33	10 ± 2	11 ± 3	24 ± 9	34 ± 1	69 ± 23
100	10 ± 4	9 ± 3	18 ± 1	25 ± 1	81 ± 15
333	8 ± 1	1 ± 2	0 ± 0	1 ± 2	40 ± 12
Sodium azide (1)	106 ± 11	n. a.	n. a.	n. a.	552 ± 55
9- AA (20)	n. a.	79 ± 45	n. a.	n. a.	n. a.
2-NF (1)	n. a.	n. a.	80 ± 15	76 ±18	n. a.
	Results with S9
DMSO	8 ± 2	14 ± 2	26 ± 2	40 ± 1	97 ± 9
1	9 ± 2	8 ± 3	23 ± 3	37 ± 10	118 ± 27
3	11 ± 3	8 ± 2	27 ± 14	43 ± 6	113 ± 13
10	12 ± 5	8 ± 5	24 ± 5	43 ± 4	106 ± 8
33	11 ± 3	14 ± 4	27 ± 2	46 ± 1	117 ± 15
100	14 ± 0	16 ± 3	12 ± 2	50 ± 10	90 ± 8
333	11 ± 2	5 ± 2	10 ± 1	23 ± 5	78 ± 7
2- AA (0.5)	n. a.	n. a.	593 ± 42	557 ± 31	893 ± 34
2- AA (1)	199 ± 2	189 ± 18	n. a.	n. a.	n. a.

 

Table 2 Results of Test II

Dose (µg/plate)	Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium
	TA 1535	TA 1537 (re tested)	TA 1538	TA 98	TA 100
	Results without S9
DMSO	14 ± 4	13 ± 2	18 ± 6	31 ± 2	98 ± 8
1	n. a.	14± 2	12± 2	27± 7	95± 12
3	14± 3	13± 3	14± 7	24± 3	90± 5
10	10± 3	10± 6	23± 4	19±2	83± 7
33	16± 4	17± 5	17±3	23± 6	78± 6
100	15± 8	14±1	25±7	21± 6	56± 6
333	12± 2	0±0	2± 1	0± 0	3± 4
1000	0± 0	n. a.	n. a.	n. a.	n. a.
Sodium azide (1)	192 ± 21	n. a.	n. a.	n. a.	493 ± 75
9- AA (20)	n. a.	426 ± 205	n. a.	n. a.	n. a.
2-NF (1)	n. a.	n. a.	211 ± 6	171 ± 20	n. a.
	Results with S9
DMSO	12± 4	8± 1	29± 8	34± 7	107± 4
3	14± 1	10± 1	37± 3	38± 2	97± 10
10	8± 2	11± 7	29± 5	Contaminated	80± 6
33	21± 2	9± 8	26± 5	30± 5	85± 5
100	6± 3	6± 3	25± 4	36± 5	74± 2
333	7± 1	0± 0	14± 2	13± 2	50± 3
1000	0± 0	0± 0	0± 0	0± 0	0± 0
2- AA (0.5)	n. a.	n. a.	458± 29	350± 37	720± 103
2- AA (1)	64± 22	56± 6	n. a.	n. a.	n. a.

 

Conclusions:

The test item is not considered to be mutagenic in S. typhimurium strains when tested in dimethylsulphoxide at concentrations extending into the toxic range.

Executive summary:

The test item was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at concentrations ranging from 1 µg to 1000 µg per plate at which concentration the substance precipitated. The tests were conducted on agar plates in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity. Concurrent positive controls demonstrated the sensitivity of the assay and the metabolizing activity of the S9 mix. The test item induced no mutagenic activity in Salmonella typhimurium, in either the presence or absence of S9 mix, in either of 2 independent experiments. Toxicity to the bacteria was observed at the higher concentrations of test item.

It is concluded that the test item is not mutagenic in S. typhimurium when tested in dimethylsulphoxide at concentrations extending into the toxic range.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-06-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 88/302/EEC Mutagenicity In vitro Mammalian Cell - Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian gene mutation assay (HPRT)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 147-270300-1

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: University of Ulm
- Doubling time: 10 - 14 h
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media:
hypoxanthine-free Eagle´s Minimal Essential Medium, supplemented with nonessential amino acids, L-glutamine (2 mM), MEM-vitamins, NaHC03, penicillin (100 units/mL), streptomycin (100 µg/mL) and heat-inactivated fetal calf serum (final concentration: 10%) (Seromed). This medium is referred to as culture medium. During treatement the serum content was reduced to 2%.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction

Test concentrations with justification for top dose:

Without metabolic activation concentrations ranging from 0.01 to 15 µg/mL
With metabolic activation concentrations were between 0.5 and 96 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: selected based on test item solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 4x10^6 per flask

DURATION
- Preincubation period: 16-24 h
- Exposure duration: 5 h
- Expression time: 6 d

SELECTION AGENT: 6-TG

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

- Mutant frequencies will only be used for assessment, if at least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10% or greater.

- A trial will be considered positive if a concentration-related and in parallel cultures reproducible increase in mutant frequencies is observed. To be relevant, the increase in mutant frequencies should be at least two to three times that of the highest negative or vehicle control value observed in the respective trial. If this result can be reproduced in a second trial, the test substance is considered to be mutagenic.

- Despite these criteria, a positive result will only be considered relevant, if no significant change in osmolality compared to the vehicle control can be observed. Otherwise, unphysiological culture conditions may be the reason for the positive result (Scott et al, 1991).

- A test substance will be judged as equivocal if there is no strictly concentration related increase in mutation frequencies but if one or more concentrations induce a reproducible and biologically relevant increase in mutant frequencies in all trials.

- An assay will be considered negative if no reproducible and relevant increases of mutant frequencies were observed.

Statistics:

The statistical analysis relies on the mutant frequencies which are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights (Hsie et al., 1981; Arlett et al., 1989).
According to the acceptance criteria mutant frequencies based on less than 5 plates and/or on a relative survival to treatment and/or a relative population growth and/or an absolute cloning efficiency below 10% are not included in the statistical analysis.

The two mutant frequency values obtained per group are, although somewhat related, considered as independent measurements thus increasing the power of the statistical tests applied. Since the protocol of the HPRT assay requires at least two independent trials, the overall analysis without respectively with activation is the most important one for classifying substances into mutagens and non-mutagens. However, separate analyses will be run for each trial in order to examine the consistency of the results.
All acceptable groups are included in the weighted analysis of variance followed by pain/vise comparisons to the vehicle control on a nominal significance level of a = 0.05 using the Dunnett test (Dunnett, 1955). The regression analysis part is performed on the basis of the actual concentrations thereby omitting the positive, negative and vehicle controls. If there is a significant concentration related increase of the mutant frequency (a = 0.05) in the main analysis the highest concentration will be dropped and the analysis will be repeated. This procedure will be repeated until p > 0.05. In that way eliminated concentrations are flagged correspondingly.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 at 1 µg/mL and above; with S9 at 5 µg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: DMSO was used as vehicle
- Precipitation: occured at concentrations of 1200 and higher

RANGE-FINDING/SCREENING STUDIES: yes

HISTORICAL CONTROL DATA: Please refer to "Any other information including tables"

Table 1: Summary of mutant frequency [10^-6] of the HPRT mutagenesis assay in V79 cells

 

Mutant Frequency [10^-6]	Without metabolic activation			With metabolic activation
Concentration [µg/mL]	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 4	Trial 5
Negative control	1.8 3.6	0.5 0.5	1.3 0.6	0.7 2.4	0.7 0.7	1.2 1.2	1.2 1.4	0.9 1.1
Vehicle control (DMSO)	3.0 3.0	0.7 0.4	2.4 2.4	1.4 1.4	1.6 2.4	0.5 0.4	1.8 4	1.5 1.0
0.01	-	0.7 0.4	-	-	-	-	-	-
0.05	-	0.4 0.8	-	-	-	-	-	-
0.1	4.8 0.5	0.4 0.4	-	0.4 0.8	-	-	-	-
0.5	3.7 0.7	1.6* 1.6	-	0.4 1.3	3.5 1.0	-	-	-
1	3.4 1.2	0.4 1.0	2.3 2.9	1.6 0.6	1.3 1.4	-	-	-
2	3.4 2.7	-	2.3 6.2	0.9 0.8	1.1 1.2	-	-	-
3	7.8 13.7	5.3* 4.6#	5.5 1.5	-	-	-	-	-
4	12.6 8.8	-	5.8 5.4	1.3 0.5	1.7 0.8	-	-	-
5	23.1 1.5	-	3.4 2.7	-	-	-	-	-
6	-	N	3.8 8.5	1.3 3.3	0.6 1.1	-	-	-
7	-	-	0.8 2.0	-	-	-	-	-
8	-	-	0.7 1.6	0.4 2.4	1.1 0.6	-	-	-
9	-	N	-	-	-	-	-	-
10	-	-	-	-	2.7 1.6	-	-	-
12	-	N	-	-	5.1 0.9	0.4 0.5	0.4 0.8	-
14	-	-	-	-	1.4 3.4	0.4 0.4	-	-
15	-	N	-	-	-	-	-	-
16	-	-	-	-	-	5.1* 3.5	-	-
18	-	-	-	-	-	0.5 0.8	0.4 1.1	-
20	-	-	-	-	-	0.4 0.7	-	-
22	-	-	-	-	-	1.2 1.2	-	-
24	-	-	-	-	-	0.4 2.8	2.2 1.1	1.6 2.0
30	-	-	-	-	-	-	1.0 0.5	-
36	-	-	-	-	-	-	0.5 0.6	1.6 0.5
42	-	-	-	-	-	-	0.4 2.4	-
48	-	-	-	-	-	-	1.0 0.5	N
60	-	-	-	-	-	-	-	N
72	-	-	-	-	-	-	-	N
84	-	-	-	-	-	-	-	N
96	-	-	-	-	-	-	N	N
Positive control (EMS)	242.7 374.2	458.6 353.6	242.7 374.2	-	-	-	-	-
Positive control (DMBA)	-	-	-	109.8 123.2	36 104.1	112.2 58.8	133.1 157.1	18.1 21.1
Historical Control (solvent: DMSO)	0 – 14.8			0.4 – 20.3
Historical Control (solvent: none)	0 – 19.3			0.4 – 17.9
# cytotoxicity (survival to treatment or population growth < 10 % of vehicle control) N not tested due to cytotoxicity * mutant frequency in each parallel culture > 2´(mutant frequency of highest control)

 

Conclusions:

The test item was tested in the V79/HPRT-test in concentrations ranging from 0.01 µg/mL to 15 µg/mL without S9 mix and from 0.1 µg/mL to 96 µg/mL with S9 mix. Under both activation conditions, clear cytotoxic effects were induced. The test item induced no biologically relevant increases in mutant frequencies. The positive controls EMS and DMBA had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant frequencies as compared to the corresponding negative controls and thus demonstrated the sensitivity of the test system and the activity of the used S9 mix. Despite this sensitivity, the test item is considered to be non-mutagenic in the V79/HPRT forward mutation assay, both with and without metabolic activation.

Executive summary:

The test item was evaluated for point mutagenic effects at the hypoxanthine-guanine phosphoribosyl transferase locus (forward mutation assay) in V79 cell cultures. Without S9 mix concentrations of up to and including 15 µg/mL were employed. With S9 mix concentrations up to and including 96 µg/mL were used. Without and with S9 mix the test item induced decreases in survival to treatment and decreases in relative population growth. These results revealed a significant concentration-related cytotoxicity of the test item. Precipitation of the test item in the culture medium was not observed. Without and with S9 mix there was no biologically relevant increase in mutant frequency above that of the vehicle controls.

Ethyl methanesulfonate and Dimethylbenzanthracene induced clear mutagenic effects and demonstrated the sensitivity of the test system and the activity of the S9 mix.

Based on these results, the test item is considered to be non-mutagenic in the V79/HPRT Forward Mutation Assay, both with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The acute oral administration of 200, 660, and 2000 mg/kg body weight of the test item to male and female mice produced no significant increases in the frequency of micronuclei in the polychromatic erythrocyte cells. Therefore, under the conditions of this study, the test item is considered not to be clastogenic at any of the levels tested.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: micronucleus assay in vivo

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York
- Age at study initiation: 32-40 d
- Assigned to test groups randomly: yes
- Housing: individually in wire-mesh cages
- Diet: ad libitum
- Water ad libitum
- Acclimation period: 19 d

ENVIRONMENTAL CONDITIONS
- Photoperiod: 12 / 12 hrs dark / hrs light
- controlled climatic conditions

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: corn oil
- Concentration of test material in vehicle: as required
- Amount of vehicle: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was freshly prepared on the day of administration. The dose solutions were prepared on a weight per volume basis. The dose levels were chosen based on the results of a range finding study.

Duration of treatment / exposure:

single application

Frequency of treatment:

see above

Post exposure period:

sampling times: 30, 48 and 72 hrs post treatment

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

660 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

15 per treatment dose and vehicle control; 5 for positive control

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw

Tissues and cell types examined:

femur bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Preliminary range-finding study

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice by cervical dislocation, bone marrow cells were collected from both femurs of each animal by aspiration into 4 mL of 1:1 fetal calf serum/phosphate buffer. The aspirate was centrifuged for five minutes at approximately 1000 rpm. The supernate was decanted and the cells were resiispended in the remaining volume. Bone marrow smears were prepared for each animal by placing one or two drops of the marrow on a microscope slide and spreading it across the slide with another slide. One slide was made for each animal and marked with the animal's identification number. Slides were allowed to air dry over night.

Slide Staining
The cells were fixed in methanol for 15 minutes and rinsed twice in distilled H2O, Fresh stain was prepared by mixing 75 mL of giemsa stain (Harleco) with 175 mL of phosphate buffer (pH 6.8). The cells were stained in this solution for 30 minutes, rinsed once in the same buffer and once in distilled H2O. Slides were air dried and subsequently mounted with glass coverslips.
Erythrocytes that stain blue or with a bluish tint were scored as polychromatic and those staining red or with a red tint were scored as normochromatic. Only cells showing good morphology were scored. Objects within a cell that appeared to be round, darkly stained and in the same focal plane as the cell were scored as micronuclei.

METHOD OF ANALYSIS:
Cytogenetic Analysis
After all slides were prepared, stained, and coverslipped, a code number was temporarily assigned to each animal by a person not involved in the scoring of the slides. The slides were not decoded until all had been analyzed. One thousand polychromatic erythrocytes (PCE) were scored from each mouse for the presence of micronuclei. Slides were analyzed with a high power oil immersion lens (100x). The number of normochromatic erythrocytes (NCE) per 1000 PCE was also recorded.

Statistics:

Upon completion of all scoring, the slides were decoded and the data were entered into the appropriate group for statistical analyses.

The mean number of micronuclei per 1000 PCE was statistically analyzed by Analysis of Variance (ANOVA) with individual group comparisons performed by Dunnett's-t-Test. The number of micronuclei per 1000 PCE for each animal was entered into the statistical program. The analysis was one-tailed at the 99% confidence interval (p < 0.01).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

2/15 animals died at 660 mg/kg bw; 9/15 died at 2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 160 - 2500 mg/kg bw
- Clinical signs of toxicity in test animals: One animal died (treatment group: 2500 mg/kg bw); Males and females of the two higher dose groups showed greater loss of body weight

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: none
- Ratio of PCE/NCE: 1.2 to 1.7:1 for control animals; approximately 0.6 to 2.1:1 for treated animals

Clinical Observations and Mortality

The number and severity of abnormal observations increased along with increased dose. Two males from Group 4 (660 mg/kg bw) and seven males and two females from Group 5 (2000 mg/kg bw) died while on study.

Body Weights

Most animals, including controls, exhibited slight body weight loss following treatment. Mice administered the highest level (2000 mg/kg bw) showed the largest decreases in body weight.

Cytogenetic Analysis

Results show that no statistically significant increase in the frequency of micronuclei compared to control values was seen in any of the groups treated with the test item.A statistically significant increase in micronucleus frequency (p = 0.0017) was seen in Group 2, the Cyclophosphamide positive control.

Ratio of Polychromatic (PCE) to Normochromatic (NCE) Erythrocytes

Ratio of PCE to NCE ranged from approximately 1.2 to 1.7:1 for control animals and from approximately 0.6 to 2.1:1 for treated animals. Although a large variance was seen between animals of the same group for the ratio of PCE:NCE, animals exposed to the highest level (2000 mg/kg bw) showed lower PCE:NCE ratios than their controls and PCE:NCE ratios decreased with increasing time in the high dose group.Since polychromatic erythrocytes (PCE) are newly formed erythrocytes, it appears that the test material may be producing toxicity in the nucleated stem cells, which would cause a decrease in the number of new erythrocytes produced.

Conclusions:

The acute oral administration of 200, 660, and 2000 mg/kg body weight of the test item to male and female mice produced no significant increases in the frequency of micronuclei in the polychromatic erythrocyte cells. Toxic effects of the test material were seen in the clinical observations and animal deaths at the the higher dose levels. Therefore, under the conditions of this study, the test item is considered not to be clastogenic at any of the levels tested.

Executive summary:

The study was designed to evaluate the clastogenic potential of the test item as measured by increases in micronuclei production in polychromatic erythrocytes from the bone marrow of Charles River, CD® -7 mice. A single dose of the test material was administered by oral gavage to four groups of 15 male and 15 female mice at levels of 0, 200, 660, and 2000 mg/kg of body weight.

 

Five males and five females from each group were scheduled to be sacrificed at 30, 48 and 72 hours after the single administration of the test material. Results show that no statistically significant increase in the frequency of micronuclei compared to control values was seen for any of the levels that were tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay in vitro

The purpose of this study was to examine the test item for mutagenic activity in the Salmonella typhimuriurn/Mammalian Microsome mutagenicity test. Five S. typhimurium strains, TA1535, TA 1537, TA1538, TA100 and TA98 were tested, both with and without mammalian S9 mix for metabolic acitvation. Concentrations of 500, 166.7, 55.6, 18.5 and 6.2 µg/plate were applied. Highest concentrations appeared to be cytotoxic in all strains tested. Based on the results of this study no mutagenic potential of the test item was observed.

Another bacterial reverse mutation assay is available. The test item was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at concentrations ranging from 1 µg to 1000 µg per plate at which concentration the substance precipitated. The tests were conducted on agar plates in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity. Concurrent positive controls demonstrated the sensitivity of the assay and the metabolizing activity of the S9 mix. The test item induced no mutagenic activity in Salmonella typhimurium, in either the presence or absence of S9 mix, in either of 2 independent experiments. Toxicity to the bacteria was observed at the higher concentrations of test item.

It is concluded that the test item is not mutagenic in S. typhimurium when tested in dimethylsulphoxide at concentrations extending into the toxic range.

Mammalian cell gene mutation assay in vitro

The test item was evaluated for point mutagenic effects at the hypoxanthine-guanine phosphoribosyl transferase locus (forward mutation assay) in V79 cell cultures. Without S9 mix concentrations of up to and including 15 µg/mL were employed. With S9 mix concentrations up to and including 96 µg/mL were used. Without and with S9 mix the test item induced decreases in survival to treatment and decreases in relative population growth. These results revealed a significant concentration-related cytotoxicity of the test item. Precipitation of the test item in the culture medium was not observed. Without and with S9 mix there was no biologically relevant increase in mutant frequency above that of the vehicle controls.

Ethyl methanesulfonate and Dimethylbenzanthracene induced clear mutagenic effects and demonstrated the sensitivity of the test system and the activity of the S9 mix.

Based on these results, the test item is considered to be non-mutagenic in the V79/HPRT Forward Mutation Assay, both with and without metabolic activation.

Unscheduled DNA Synthesis (UDS) assay in vitro

The test item was tested for its ability to induce unscheduled DNA synthesis (UDS) in primary cultures of adult rat hepatocytes (HPCs) as measured by silver grain counts in photographic emulsion formed by radiation from [63H]-thymidine taken up by the cells.Cultures were established with cells derived from the collagenase-perfused liver of young, adult, male, Fischer 344 rats. Eight different concentrations of the test item from 3 µg/mL to 13.5 µg/mL were tested. In neither of 2 independent experiments did any tested concentration of the test item meet any of the 3 criteria indicative of unscheduled DNA synthesis induction.

It is concluded that the test item does not induce unscheduled DNA synthesis in primary cultures of rat hepatocytes, when tested in dimethylsulphoxide at concentrations which extended into the toxic range.

Micronucleus assay in mice in vivo

The study was designed to evaluate the clastogenic potential of the test item as measured by increases in micronuclei production in polychromatic erythrocytes from the bone marrow of Charles River, CD® -7 mice. A single dose of the test material was administered by oral gavage to four groups of 15 male and 15 female mice at levels of 0, 200, 660, and 2000 mg/kg of body weight.

Five males and five females from each group were scheduled to be sacrificed at 30, 48 and 72 hours after the single administration of the test material. Results show that no statistically significant increase in the frequency of micronuclei compared to control values was seen for any of the levels that were tested.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity in vitro and in vivo the test item is not classified as genotoxic according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.