3-iodo-2-propynyl butylcarbamate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

no

Test guideline 3

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)

Test material

Test material information

Constituent 1

Reference substance name:

3-iodo-2-propynyl butylcarbamate

EC Number:

259-627-5

EC Name:

3-iodo-2-propynyl butylcarbamate

Cas Number:

55406-53-6

Molecular formula:

C8H12INO2

IUPAC Name:

3-iodoprop-2-yn-1-yl butylcarbamate

Specific details on test material used for the study:

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

Method

Target gene:

HPRT locus

Species / strain

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

Controls

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.

Results and discussion

Test results

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"

Any other information on results incl. 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)

 

Applicant's summary and conclusion

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.