Iodomethane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 January 2001 to 26 January 2001

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

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Iodomethane

EC Number:

200-819-5

EC Name:

Iodomethane

Cas Number:

74-88-4

Molecular formula:

CH3I

IUPAC Name:

iodomethane

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Iodomethane
- Physical state: liquid
- Analytical purity: 99.7%
- Impurities (identity and concentrations): 0.2 % water and <0.1 % methanol
- Composition of test material, percentage of components: not stated
- Isomers composition: not stated

Method

Target gene:

S. typhimurium: Histidine locus
E. coli: Tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored.

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9

Test concentrations with justification for top dose:

- Initial toxicity-mutation assay: 5000, 1500, 500, 150, 50, 15, 5.0, 1.5, 0.50, 0.15, 0.050 and 0.015 μg/plate.
- Toxicity was observed at 5000 μg/plate with most test conditions. No precipitate was observed. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 μg/plate.
- Confirmatory mutagenicity assay: 5000, 1500, 500, 150, 50 and 15 μg/ plate.

Vehicle / solvent:

- Vehicle used: sterile distilled water
- Justification for choice of solvent/vehicle: Water was selected as the solvent of choice based on the Sponsor's request, compatibility with the target cells and solubility of the test material.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

AGAR: On the day of its use, minimal top agar, containing 0.8 % agar (w/v) and 0.5 % NaCl (w/v), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionised water produced by the Milli-Q Reagent Water System. Bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (w/v) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (w/v) agar and supplemented with 2.5 % (w/v) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (w/v) Oxoid Nutrient Broth No. 2 (dry powder).

PLATING
- In the initial toxicity-mutation assay, two sets of test material dilutions were prepared immediately before use, one set was used for plating without S9 activation and the other set was used for plating with S9 activation. In the confirmatory mutagenicity assay, two sets of test material dilutions were prepared immediately before use, one set of dilutions for dose levels 5000, 1500 and 500 μg/ plate in the presence and absence of S9 activation and the other set of test material dilutions for 150, 50 and 15 μg/plate in the presence and absence of S9 activation. 0.5 mL of S9 or sham mix, 100 μL of tester strain and 500 μL of vehicle or test material were added to 13 X 100 mm glass culture tubes with Teflon®-lined screw caps pre-heated to 37 ± 2 °C. The tubes receiving test material were capped during the preincubation period. After vortexing, these mixtures were incubated with shaking for 60 ± 2 minutes at 37 ± 2 °C.
- Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test material aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37 ± 2 °C. For the confirmatory mutagenicity assay, the test material plates were placed in desiccators by dose level for 24 hours and then removed from the desiccator for the remainder of the incubation period. Plates that were not counted immediately following the incubation period were stored at 2 to 8 °C until colony counting could be conducted.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- The condition of the bacterial background lawn was evaluated for evidence of test material toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown below.
1: Normal- Distinguished by a healthy microcolony lawn.
2: Slightly Reduced- Distinguished by a noticeable thinning of the microcolony lawn and possibly a slight increase in the size of the microcolonies compared to the vehicle control plate.
3: Moderately Reduced- Distinguished by a marked thinning of the microcolony lawn resulting in a pronounced increase in the size of the microcolonies compared to the vehicle control plate.
4: Extremely Reduced- Distinguished by an extreme thinning of the microcolony lawn resulting in an increase in the size of the microcolonies compared to the vehicle control plate such that the microcolony lawn is visible to the unaided eye as isolated colonies.
5: Absent- Distinguished by a complete lack of any microcolony lawn over ≥ 90 % of the plate.
6: Obscured by Precipitate- The background bacterial lawn cannot be accurately evaluated due to microscopic test material precipitate.
NP: Non-Interfering Precipitate- Distinguished by precipitate on the plate that is visible to the naked eye but any precipitate materials detected by the automated colony counter total less than 10 % of the revertant colony count (e.g., < 3 materials on a plate with 30 revertants.)
IP: Interfering Precipitate- Distinguished by precipitate on the plate that is visible to the naked eye and any precipitate pmaterials detected by the automated colony counter exceed 10 % of the revertant colony count (e.g., > 3 materials on a plate with 30 revertants.)

- Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually.

CRITERIA FOR A VALID TEST
The following criteria must be met for the mutagenicity assay to be considered valid:
- All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- Cultures of tester strains T A98 and TA 100 must demonstrate the presence of the pKM1O1 plasmid R-factor.
- All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
- All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 to 50; TA100, 80 to 240; TA1535, 5 to 45; TA1537, 3 to 21; WP2 uvrA, 10 to 60.
- To ensure that appropriate numbers of bacteria are plated, tester strain culture titres must be greater than or equal to 0.3 x 10^9 cells/mL.
- The mean of each positive control must exhibit at least a three-fold increase in the number of revertants over the mean value of the respective vehicle control.
- A minimum of three non-toxic dose levels are required to evaluate assay data.
- A dose level is considered toxic if one or both of the following criteria are met: (1) A > 50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A reduction in the background lawn.

Evaluation criteria:

EVALUATION OF RESULTS
- For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. For the test material to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test material.
- Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value.
- Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

INITIAL TOXICITY MUTATION ASSAY
- In the initial toxicity -mutation assay, the maximum dose tested was 5000 μg/plate; this dose was achieved using a concentration of 10 mg/mL and a 500 μL plating aliquot. Concentrations from 30 pg/mL to 10 mg/mL were soluble and clear solutions.
- The dose levels tested were 5000, 1500, 500, 150, 50, 15, 5.0, 1.5, 0.50, 0.15, 0.050 and 0.015 μg/plate. Toxicity was observed at 5000 μg/plate with most test conditions. No precipitate was observed. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 μg/plate. In Experiment B1 (Initial Toxicity-Mutation Assay), no positive responses were observed with any of the tester strains in the presence and absence of S9 activation.
 
CONFIRMATORY MUTATION ASSAY
- The dose levels tested were 5000, 1500, 500, 150, 50 and 15 μg/plate. Toxicity was observed at 5000 μg/ plate with most test conditions. No precipitate was observed.
- In Experiment B2 (Confirmatory Mutagenicity Assay), no positive responses were observed with any of the tester strains in the presence and absence of S9 activation.
- All criteria for a valid study were met.

Any other information on results incl. tables

Table 1: Summary of Experiment B1 (INITIAL TOXICITY MUTATION ASSAY)

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 0.015 0.050 0.15 0.50 1.5 5.0 15 50 150 500 1500 5000	122 105 118 90 116 108 100 117 109 115 130 98 89	9 6 13 11 13 11 14 8 15 9 10 11 8	10 8 10 9 10 13 10 6 10 10 8 4 7	13 7 11 13 15 12 14 11 15 11 11 14 11	7 6 6 4 7 6 3 3 6 5 4 7 3
+	Solvent 0.015 0.050 0.15 0.50 1.5 5.0 15 50 150 500 1500 5000	119 109 141 115 125 117 108 104 128 107 149 127 116	17 20 16 14 12 19 16 17 10 15 15 7 12	11 9 10 9 8 9 9 8 11 7 8 11 5	26 23 19 20 22 19 16 19 17 21 26 25 21	5 7 4 4 5 4 5 7 6 6 6 6 6
Positive Controls
-	Name	SA	SA	MMS	2NF	9AA
	Concentration (µg/plate)	1.0	1.0	1000	1.0	75
	Mean no. colonies/plate	414	353	296	607	1116
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1.0	1.0	10	1.0	1.0
	Mean no. colonies/plate	672	68	166	607	80

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

MMS = Methyl methanesulfonate

 

 

Table 2: Summary of Experiment B2 (CONFIRMATORY MUTATION ASSAY)

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 15 50 150 500 1500 5000	89 81 79 82 104 99 80	9 8 8 12 14 9 4	12 9 11 14 10 14 8	13 11 10 14 10 11 2	5 4 5 5 5 5 2
+	Solvent 15 50 150 500 1500 5000	89 81 95 104 106 109 72	15 13 11 10 7 13 8	12 12 12 15 12 11 9	20 13 17 17 16 15 1	5 8 6 6 5 6 2
Positive Controls
-	Name	SA	SA	MMS	2NF	9AA
	Concentration (µg/plate)	1.0	1.0	1000	1.0	75
	Mean no. colonies/plate	566	559	376	572	1106
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1.0	1.0	10	1.0	1.0
	Mean no. colonies/plate	586	119	216	679	66

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

MMS = Methyl methanesulfonate

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Executive summary:

The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted in accordance with the standardised guideline EPA 84-2, under GLP conditions.

The test material was tested in the Bacterial Reverse Mutation Assay (Ames) using Salmonella typhimurium tester strains A98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the preincubation method. The first phase, the preliminary toxicity-mutation assay, was used to establish the dose-range for the mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test material. Water was selected as the solvent of choice based on the Sponsor's request, compatibility with the target cells and solubility of the test material.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg/plate; this dose was achieved using a concentration of 10 mg/mL and a 500 μL plating aliquot. Concentrations from 30 pg/mL to 10 mg/mL were soluble and clear solutions. The dose levels tested were 5000, 1500, 500, 150, 50, 15, 5.0, 1.5, 0.50, 0.15, 0.050 and 0.015 μg/plate. Toxicity was observed at 5000 μg/plate with most test conditions. No precipitate was observed. Based on the findings of the toxicity-mutation assay, the maximum dose plated in the mutagenicity assay was 5000 μg/plate.

In the confirmatory mutagenicity assay, no positive response was observed. The dose levels tested were 5000, 1500, 500, 150, 50 and 15 μg/ plate. Toxicity was observed at 5000 μg/plate with most test conditions. No precipitate was observed.

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.