Iodomethane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

IN VITRO STUDIES

Ames

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 study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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.

Mammalian cell mutation (CHO/HGPRT Assay)

The genetic toxicity of the test material was investigated in accordance with the standardised guideline EPA 84-2, under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested in the CHO/HGPRT Mutation Assay in the absence and presence of Aroclor-induced rat liver S9.

The preliminary toxicity assay was used to establish the dose range for the mutagenesis assay. The mutagenesis assays was used to evaluate the mutagenic potential of the test material. Sterile distilled water was chosen by the Sponsor as the solvent for the test material. The test material was soluble in sterile distilled water at a concentration of 14.3 mg/mL, the maximum concentration prepared.

In the preliminary toxicity assay, the maximum concentration tested was 1430 μg/mL (10 mM). There was no visible precipitate in the treatment medium at any concentration. Selection of dose levels for the mutagenesis assay was based on the cloning efficiency relative to the solvent control. Substantial toxicity, i.e., cloning efficiency < 50 % of the solvent control, was observed at≥505 μg/mL with and without S9 activation. Based on these findings, the doses chosen for the mutagenesis assay ranged from 100 to 600 μg/mL for the nonactivated cultures and 100 to 500 μg/mL for the S9-activated cultures.

In the mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies > 40 mutants per 10^6 clonable cells, were observed. No visible precipitate was observed in treatment medium at any concentration. Toxicity, i.e., cloning efficiency≤50 % of the solvent control, was observed at doses of≥125 μg/mL without activation and≥150 μg/mL with activation. 

In the presence of S9 RCE reduced to 41%, not considered sufficient in accordance with the guideline requirements (10-20%) RCE reduced to 25%. Whilst not considered acceptable with guidance (cytotoxicity 10-20%), in the absence of a dose related increase in mutant frequency, a reduction in RCE to 25% was deemed acceptable

However, in the absence of S9 mutant frequency was not determined due to contamination in the mutation plates. Instead a dose of 125 mg/mL (the next highest concentrations) was assessed for mutant frequency, where RCE was reduced to 25 %. In the absence of any dose related increase in mutant frequency up RCE of 25 % this level of toxicity was deemed acceptable. In the presence of S9 RCE was reduced to 41 % at a concentration of 200 µg/mL where mutant frequency was determined.

Under the conditions of this study, the test material was concluded to be negative in the CHO/HGPRT Mutation Assay.

Chromosome aberration test

The genetic toxicity of the test material was investigated in accordance with the standardised guideline EPA 84-2, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test material.

Water was determined to be the solvent of choice based on information provided by the Sponsor, the solubility of the test material, and compatibility with the target cells. The test material was soluble in water at a concentration of 14.2 mg/mL, the maximum concentration tested.

In the preliminary toxicity assay, the maximum dose tested was 1420 μg/mL. The test material was soluble in treatment medium at all dose levels tested. Selection of dose levels for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Substantial toxicity, i.e., at least 50 % cell growth inhibition, was observed at dose levels of 426 μg/mL in both the non-activated and S9 activated 4 hour exposure groups, and in the non-activated 20 hour continuous exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 50 to 350 μg/mL for the nonactivated 4 hour exposure group, and ranged from 25 to 350 μg/mL for the S9 activated 4 hour exposure group. The doses chosen for the non-activated 20 hour continuous exposure group of the chromosome aberration assay ranged from 25 to 250 μg/mL. In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the nonactivated test system and for 4 hours in the S9 activated test system, and all cells were harvested at 20 hours after treatment initiation. The test material was soluble in treatment medium at all dose levels tested.

Selection of doses for microscopic analysis was based on toxicity and mitotic index reduction (the lowest dose with approximately 50 % reduction in cell growth and with sufficient scorable metaphase cells and two lower doses) in all harvests.

Under the conditions of this study, the test material was concluded to be positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in Chinese hamster ovary (CHO) cells.

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:

09 January 2001 to 26 January 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

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

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 / 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.

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.

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

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 January 2001 and 26 February 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

other: chromosome aberration study in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 20.
- The freeze lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination.
- This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced

Test concentrations with justification for top dose:

- Preliminary toxicity test: 4 hour -/+S9 and 20 hour –S9: 0.142, 0.426, 1.42, 4.26, 14.2, 42.6, 142, 426, 1420 µg/mL
- The selection of dose levels for analysis of chromosome aberrations in CHO cells was based upon toxicity of the test article in treatment medium. The highest dose level selected for evaluation was the dose which induced approximately 50 % toxicity, as measured by cell growth inhibition, relative to the solvent control, with a sufficient number of scorable metaphase cells. Two additional lower dose levels were included in the evaluation. Based upon the results of the toxicity study, the dose levels selected for testing in the chromosome aberration assay were as follows:
4 hour -S9: 50, 100, 150, 200, 250, 300 and 350 µg/mL
20 hour -S9: 25, 50, 100, 150, 175, 200 and 250 µg/mL
4 hour +S9: 25, 50, 100, 150, 200, 250, 300 and 350 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: Water was determined to be the solvent of choice based on information provided by the Sponsor, the solubility of the test material, and compatibility with the target cells. The test material was soluble in water at a concentration of 14.2 mg/mL, the maximum concentration tested.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

PRELIMINARY TOXICITY ASSAY
- CHO cells were seeded for each treatment condition at approximately 5 x 10^5 cells/25 cm^2 flask and were incubated at 37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air for 16 to 24 hours. Treatment was carried out by refeeding the flasks with 4.5 mL complete medium (McCoy's SA medium supplemented with 10 % foetal bovine serum (FBS), 100 units penicillin and 100 μg streptomycin/mL, and 2 mM L-glutamine) for the non-activated study or S9 reaction mixture (3.5 mL serum-free medium plus 1 mL of 5X S9 mix) for the activated study, to which was added 500 μL dosing solution of test material in solvent or solvent alone.
- The osmolality of the highest concentration of dosing solution in the treatment medium was measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape.
- The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9. At completion of the 4 hour exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline (CMF-PBS), refed with 5 mL complete medium and returned to the incubator for a total of 20 hours from the initiation of treatment. At 20 hours after the initiation of treatment the cells were harvested by trypsinisation and counted using a Coulter counter. The presence of test material precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control.

CHROMOSOME ABERRATION ASSAY
- The chromosome aberration assay was performed using standard procedures as in the preliminary toxicity test, by exposing duplicate cultures of CHO cells to the test material as well as positive and solvent controls.
- In the non-activated study, the cells were exposed to the test material for 4 hours or continuously for 20 hours up to the cell harvest at 37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air. In the 4 hour exposure group, after the exposure period, the treatment medium was removed, the cells washed with CMF-PBS, refed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid® was added to duplicate flasks for each treatment condition at a final concentration of 0.1 μg/mL and the flasks returned to the incubator until cell collection.
- In the S9 activated study, the cells were exposed for 4 hours at 37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air. After the exposure period, the treatment medium was removed, the cells washed with CMF-PBS, refed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid® was added to duplicate flasks for each treatment condition at a final concentration of 0.1 μg/mL and the flasks were returned to the incubator until cell collection.
- A concurrent toxicity test was conducted in both the non-activated and the S9 activated test systems. After cell harvest an aliquot of the cell suspension was removed from each culture and counted using a Coulter counter. The presence of test material precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control.

COLLECTION OF METAPHASE CELLS
- Two hours after the addition of Colcemid®, metaphase cells were harvested for both the non-activated and S9 activated studies by trypsinisation. Cells were collected approximately 20 hours after initiation of treatment. The cells were collected by centrifugation at approximately 800 rpm for 5 minutes. The cell pellet was resuspended in 2 to 4 mL 0.075 M potassium chloride (KCl) and allowed to stand at room temperature for 4 to 8 minutes. The cells were collected by centrifugation, the supernatant aspirated and the cells fixed with two washes of approximately 2 mL Carnoy's fixative (methanol: glacial acetic acid, 3:1, v/v). The cells were stored overnight or longer in fixative at approximately 2 to 8 °C.

SLIDE PREPARATION
- To prepare slides, the fixed cells were centrifuged at approximately 800 rpm for 5 minutes, the supernatant was aspirated, and 1 mL fresh fixative was added. After additional centrifugation (at approximately 800 rpm for 5 minutes) the supernatant fluid was decanted and the cells resuspended to opalescence in fresh fixative. A sufficient amount of cell suspension was dropped onto the centre of a glass slide and allowed to air dry. The dried slides were stained with 5 % Giemsa, air dried and permanently mounted.

EVALUATION OF METAPHASE CELLS
- Slides were coded using random numbers by an individual not involved with the scoring process. To ensure that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group.
- Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Initially, the non-activated and S9 activated 4 hour exposure groups were evaluated for chromosome aberrations and if a positive result was obtained in the non-activated 4 hour exposure group, the non-activated 20 hour continuous exposure group was not necessarily evaluated for chromosome aberrations.
- Whenever possible, a minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. The number of metaphase spreads that were examined and scored per duplicate flask may have been reduced if the percentage of aberrant cells reaches a statistically significant level before 100 cells are scored.
- Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements.
- Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings.
- Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome).
- Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverised chromosome(s), pulverised cells and severely damaged cells (≥ 10 aberrations) were also recorded. Chromatid and isochromatid gaps
were recorded but not included in the analysis. The XY coordinates for each cell with chromosomal aberrations were recorded using a calibrated microscope stage. Polyploid and endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored.

Evaluation criteria:

Evaluation of Test Results
- The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies. The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell were calculated and reported for each group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.

Criteria for a Valid Test
- The frequency of cells with structural chromosome aberrations in the solvent control must be within the range of the historical solvent control.
- The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p ≤ 0.05 Fisher's exact test) relative to the solvent control.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test material dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY ASSAY
- The test material was soluble in treatment medium at all dose levels tested.
- The osmolality in treatment medium of the highest concentration tested, 1420 μg/mL, was 267 mmol/kg. The osmolality of the solvent (water) in treatment medium was 266 mmol/kg.
- The pH of the highest concentration of test material in treatment medium was approximately 7.
- Cell growth inhibition relative to the solvent control was 100 % at 1420 μg/mL, the highest concentration tested in both the non-activated 4 and 20 hour exposure groups and in the S9 activated 4 hour exposure group.

CHROMOSOME ABERRATION ASSAY
- In the chromosome aberration assay, the test material was soluble in treatment medium at all dose levels tested.
- The osmolality in treatment medium of the highest concentration tested, 3 50 μg/mL, was 270 mmol/kg. The osmolality of the solvent (water) in treatment medium was 265 mmol/kg.
- The pH of the highest concentration of test material in treatment medium was approximately 7.
- Toxicity of the test material ( cell growth inhibition relative to the solvent control) in CHO cells when treated for 4 hours in the absence of S9 activation was 50 % at 250 μg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 250 μg/mL, was 52 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 50, 150, and 250 μg/mL. The percentage of cells with structural aberrations in the test material-treated groups was significantly increased above that of the solvent control at dose levels 150 and 250 μg/mL (p ≤ 0.01, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p ≤ 0.05). The percentage ofcells with numerical aberrations in the test material-treated groups was significantly increased above that of the solvent control at dose level 150 μg/mL (p ≤ 0.05, Fisher's. exact test). The Cochran-Armitage test was also positive for a dose response (p ≤ 0.05). However, the percentage of cells with numerical aberrations (3.0 %) was within the historical solvent control range of 0 to 7.5 %. Therefore, it is not considered biologically significant. The percentage of structurally damaged cells in the MMC group was found to be statistically significant (16.5 %).
- Toxicity of the test material (cell growth inhibition relative to the solvent control) in CHO cells when treated for 4 hours in the presence of S9 activation was 40 % at 200 μg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 200 μg/mL, was 77 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 25, 100, and 200 μg/mL. The percentage of cells with structural aberrations in the test material-treated groups was statistically increased above that of the solvent control at dose levels 100 and 200 μg/mL (p ≤ 0.05 and 0.01, respectively, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p ≤ 0.05). The percentage of cells with numerical aberrations in the test material-treated groups was not significantly increased above that of the solvent control (p > 0.05, Fisher's exact test). The percentage of structurally damaged cells in the CP group was found to be statistically significant (15.0 %).
- Although the non-activated 4 hour exposure group exhibited a positive response for the induction of structural aberrations, slides from the non-activated 20 hour exposure group were evaluated for chromosome aberrations at the request of the Sponsor. Toxicity of the test material (cell growth inhibition relative to the solvent control) was 47 % at 250 μg/mL, the highest test concentration evaluated for chromosome aberrations in the nonactivated 20 hour continuous exposure group. The mitotic index at the highest dose level evaluated for chromosome aberrations, 250 μg/mL, was 53 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 50, 150, and 250 μg/mL. The percentage of cells with structural aberrations in the test material-treated groups was significantly increased above that of the solvent control at dose levels 50, 150 and 250 μg/mL (p ≤ 0.1, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p ≤ 0.05). The percentage of cells with numerical aberrations in the test material-treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of structurally damaged cells in the MMC group was found to be statistically significant (14.0 %).
- The positive and solvent controls fulfilled the requirements for a valid test.

Table 1: Summary of results

Treatment	S9 activation	Treatment time	Mean mitotic index	Cells scored	Aberrations per cell (Mean ± SD)	Cells with aberrations (%)
						Numerical	Structural
Water	-	4	9.3	200	0.000 ± 0.000	0.0	0.0
Test material 50 µg/mL	-	4	6.2	200	0.010 ± 0.100	0.5	1.0
Test material 150 µg/mL	-	4	5.4	200	0.160 ± 0.817	3.0*	8.0**
Test material 250 µg/mL	-	4	4.5	200	0.260 ± 1.162	2.0	11.0**
MMC 0.2 µg/mL	-	4	5.3	200	0.225 ± 0.553	0.0	16.5**
Water	+	4	9.2	200	0.005 ± 0.071	6.0	0.5
Test material 25 µg/mL	+	4	7.9	200	0.015 ± 0.122	8.5	1.5
Test material 100 µg/mL	+	4	7.7	200	0.045 ± 0.231	7.5	4.0*
Test material 200 µg/mL	+	4	2.1	200	0.335 ± 0.909	2.5	18.0**
CP 10 µg/mL	+	4	4.2	200	0.230 ± 0.685	5.5	15.0**
Water	-	20	6.8	200	0.000 ± 0.000	1.5	0.0
Test material 50 µg/mL	-	20	5.9	200	0.095 ± 0.741	6.0	4.5**
Test material 150 µg/mL	-	20	5.0	200	0.220 ± 0.852	1.5	14.0**
Test material 250 µg/mL	-	20	3.2	200	0.345 ± 1.167	2.0	17.5**
MMC 0.1 µg/mL	-	20	4.4	200	0.305 ± 1.281	1.0	14.0**

Treatment: Cells from all treatment conditions were harvested at 20 hours after the initiation of the treatments.

Aberrations per Cell: Severely damaged cells were counted as 10 aberrations.

Percent Aberrant Cells: *= p≤0.05; **=p≤0.01; using the Fisher’s exact test.

Conclusions:

Under the conditions of this study, the test material was concluded to be positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in Chinese hamster ovary (CHO) cells.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guideline EPA 84-2, under GLP conditions.

The test material, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test material.

Water was determined to be the solvent of choice based on information provided by the Sponsor, the solubility of the test material, and compatibility with the target cells. The test material was soluble in water at a concentration of 14.2 mg/mL, the maximum concentration tested.

In the preliminary toxicity assay, the maximum dose tested was 1420 μg/mL. The test material was soluble in treatment medium at all dose levels tested. Selection of dose levels for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Substantial toxicity, i.e., at least 50 % cell growth inhibition, was observed at dose levels of 426 μg/mL in both the non-activated and S9 activated 4 hour exposure groups, and in the non-activated 20 hour continuous exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 50 to 350 μg/mL for the nonactivated 4 hour exposure group, and ranged from 25 to 350 μg/mL for the S9 activated 4 hour exposure group. The doses chosen for the non-activated 20 hour continuous exposure group of the chromosome aberration assay ranged from 25 to 250 μg/mL. In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the nonactivated test system and for 4 hours in the S9 activated test system, and all cells were harvested at 20 hours after treatment initiation. The test material was soluble in treatment medium at all dose levels tested.

Selection of doses for microscopic analysis was based on toxicity and mitotic index reduction (the lowest dose with approximately 50 % reduction in cell growth and with sufficient scorable metaphase cells and two lower doses) in all harvests.

Under the conditions of this study, the test material was concluded to be positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in Chinese hamster ovary (CHO) cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 December 2000 and 19 March 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

yes

Remarks:

. Insufficient level of toxicity induced in the +S9 condition.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

Hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1-BH4

Details on mammalian cell type (if applicable):

- Type and identity of media: Serum-free Ham's F12 without hypoxanthine, supplemented with 5 % foetal bovine serum, 100 units penicillin/mL, 100 ug streptomycin/mL and 2 mM L-glutamine/mL
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: The freeze lot of cells was tested and found to be free of mycoplasma contamination.
- Periodically checked for karyotype stability: Cells used in the mutation assay were within four subpassages from frozen stock in order to assure karyotypic stability.
- CHO cells were cleansed in medium supplemented with hypoxanthine, arninopterin and
thymidine (HA 1) then frozen.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254

Test concentrations with justification for top dose:

- Preliminary toxicity test: 0.15, 0.5, 1.5, 5, 15, 51, 152, 505 and 1403 µg/mL
- Based on the results of the toxicity test, the doses chosen for the mutagenesis assay ranged from 100 to 600 μg/mL for the non-activated cultures and from 100 to 500 μg/mL for the S9-activated cultures. The first trial of the mutagenesis assay failed due to excessive toxicity. The second trial of the S9-activated portion failed due to contamination. The data from those assays were recorded but not reported. In the reported assays, cultures were treated with 25 to 200 μg/mL both with and without activation.
- Mutation assay: -S9: 25, 50, 100, 125, 150 and 175 µg/mL, +S9: 25, 50, 100, 150, 175 and 200 µg/mL

Vehicle / solvent:

- Vehicle used: sterile distilled water
- Justification for choice of solvent/vehicle: Sterile distilled water was chosen by the Sponsor as the solvent for the test material. The test material was soluble in sterile distilled water at a concentration of 14.3 mg/mL, the maximum concentration prepared.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

PRELIMINARY TOXICITY ASSAY
- The preliminary toxicity assay was used to establish the optimal dose levels for the mutagenesis assay and consisted of evaluation of test article effect on colony-forming efficiency. CHO cells were exposed for 5 hours at 37 ± 1 °C to the vehicle alone and nine concentrations of test article ranging from 0.15 to 1430 μg/mL in both the absence and presence of S9-activation.

MUTAGENICITY ASSAY
- The mutagenesis assay was used to evaluate the mutagenic potential of the test article. CHO cells were exposed for 5 hours at 37 ± 1 °C to the vehicle alone, appropriate positive controls and at least five concentrations of test article in duplicate in both the absence and presence of S9.

TREATMENT
- Exponentially growing CHO-K1-BH4 cells were seeded in F12FBS5-Hx at a density of 5x10^5 cells/25 cm^2 flask and were incubated at 37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2, in air for 18 to24 hours. F12FBS5-Hx is Ham's F12 medium without hypoxanthine supplemented with 5 % dialysed FBS, 100 units penicillin/mL, 100 μg streptomycin/mL and 2mM L-glutamine/mL.
- The time of initiation of chemical treatment was designated as day 0. Treatment was carried out by refeeding the treatment flasks with 4.5 mL F12FBS5-Hx for the non-activated study, or 3.5 mL F12FBS5-Hx and 1 mL S9 reaction mixture for the S9-activated study, to which was added 500 μL dosing solution of test article in vehicle or vehicle alone per 25 cm^2 flask.
- Positive control cultures were treated with 50 μL in 5 mL per 25 cm^2.
- Duplicate flasks of cells were exposed to at least five concentrations of the test article for 5 hours at 37 ± 1 °C. Due to the volatility of the test article, cultures were gassed with 5 % CO, and capped tightly prior to incubation.
- After the treatment period, all media were aspirated, the cells washed with Ca++- and Mg++- free Hanks' balanced salt solution (CMF-HBSS) and cultured in F12FBS5-Hx for an additional 18 to 24 hours at 37 ± 1 °C. At this time, the cells were subcultured to assess cytotoxicity and to initiate the phenotypic expression period.

EVALUATION OF CYTOTOXICITY
- For evaluation of cytotoxicity, the replicates from each treatment condition were detached using trypsin and subcultured independently in F12FBS5-Hx, in triplicate, at a density of 100 cells/60 mm dish. After 7 to 10 days incubation, the colonies were rinsed with HBSS, fixed with methanol, stained with 10 % aqueous Giemsa, counted and cloning efficiency determined.

EXPRESSION AND SELECTION OF THE MUTANT PHENOTYPE
- For expression of the mutant phenotype, the replicates from each treatment condition were trypsinised and subcultured independently in F12FBS5-Hx, in duplicate, at a density no greater than 10^6 cells/100 mm dish. Subculturing by trypsinising at 2 to 3 day intervals was employed for the 7 to 9 day expression period. At the end of the expression period, selection for the mutant phenotype was performed.
- For selection of the TG-resistant phenotype, the replicates from each treatment condition were trypsinised and replated, in quintuplicate, at a density of 2x10^5 cells/100 mm dish in F12FBS5-Hx containing 10 μM 6-thioguanine (TG, 2-arnino-6-mercaptopurine). For cloning efficiency determinations at the time of selection, 100 cells/60 mm dish were plated in triplicate. After 7-10 days of incubation, the colonies were fixed, stained and counted for both cloning efficiency and mutant selection.

CRITERIA FOR A VALID TEST
- The cloning efficiency of the solvent control must be greater than 50 %.
- The spontaneous mutant frequency in the solvent control must fall within the range of 0 to 25 mutants per 10^6 clonable cells.
- The positive control must induce a mutant frequency at least three times that of the solvent control and must exceed 40 mutants per 10^6 clonable cells.
- There must be at least four analysable test material concentrations with mutant frequency data.

Evaluation criteria:

- The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control (relative cloning efficiency). The mutant frequency (MF) for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selected (usually 2x 10^6 cells: 10 plates at 2 x 10^5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection, and is expressed as TG-resistant mutants per 10^6 clonable cells. For experimental conditions in which no mutant colonies were observed, mutant frequencies were expressed as less than the frequency obtained with one mutant colony. Mutant frequencies generated from doses giving < 10 % relative survival are presented in the data but were not considered as valid data points.
Because spontaneous mutant frequencies are very low for the CHO/HGPRT assay, calculation of mutagenic response in terms of fold increase in mutant frequency above the background rate does not provide a reliable indication of the significance of the observed response. The wide acceptable range in spontaneous mutant frequency also suggests the need to set a minimum mutant frequency for a response to be considered positive. In this laboratory, a more conservative approach is used which sets the minimum level at > 40 mutants per 10^6 clonable cells.
- All conclusions were based on sound scientific judgement; however, the following criteria are presented as a guide to interpretation of the data: The test material was considered to induce a positive response if there was a concentration related increase in mutant frequencies with at least two consecutive doses showing mutant frequencies of > 40 mutants per 10^6 clonable cells, if a single point above 40 mutants per 10^6 clonable cells was observed at the highest dose, the assay was considered suspect and if no culture exhibited a mutant frequency of > 40 mutants per 10^6 clonable cells, the test material was considered negative.

Statistics:

No statistical analysis was conducted.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

PRELIMINARY TOXICITY ASSAY
- CHO cells were exposed to solvent alone and nine concentrations of test material ranging from 0.15 to 1430 μg/mL (10 mM) in the absence and presence of S9 reaction mixture.
- No test material precipitate was observed at any dose level in treatment medium.
- The osmolality of the solvent control was 265 mmol/kg and the osmolality of the highest dose, 1430 μg/mL, was 279 mmol/kg.
- Cloning efficiency relative to the solvent controls (RCE) was 1 % at 1430 μg/mL without activation and 0 % at 1430 μg/mL with S9 activation. Based on the results of the toxicity test, the doses chosen for the mutagenesis assay ranged from 100 to 600 μg/mL for the non-activated cultures and from 100 to 500 μg/mL for the S9-activated cultures.

MUTAGENICITY ASSAY
- The first trial of the mutagenesis assay failed due to excessive toxicity. The second trial of the S9-activated portion failed due to contamination. The data from those assays were recorded but not reported.
- In the reported assays, cultures were treated with 25 to 200 μg/mL both with and without activation. In the non-activated system cultures treated with concentrations of 25, 50, 100, 125, 150 and 175 μg/mL were cloned for concurrent cytotoxicity. Due to toxicity at 175 μg/mL and contamination (noted at the time of selection) at 150 μg/mL, only non-activated cultures for the 25 to 125 μg/mL dose levels were seeded for mutant selection. In the S9-activated system, cultures treated with concentrations of 25, 50, 100, 150, 175 and 200 μg/mL were cloned. No test material precipitate was observed at any dose level in treatment medium.
- Relative cloning efficiency was 19 and 41 % at the highest dose tested in the non-activated and S9-activated systems, respectively. None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 10^6 clonable cells.
- All criteria for a valid study were met.

Table 1: Summary of results

Concentration (µg/mL)	-S9						+ S9
	Viability plate		Mutant plate				Viability plate		Mutant plate
	Total CE	RCE	Total Clones	CE	Total colonies	Mutants /16^6	Total CE	RCE	Total Clones	CE	Total colonies	Mutants /16^6
Solvent	0.60	100	513	0.86	2	1.3	0.72	100	438	0.73	21	14.4
25	0.67	112	337	0.67	25	18.5	0.79	110	455	0.76	15	9.9
50	0.59	99	356	0.71	13	9.1	0.62	86	457	0.76	11	7.2
100	0.43	72	384	0.64	5	3.9	0.56	77	452	0.75	9	6.0
125	0.15	25	367	0.61	8	6.5	-	-	-	-	-	-
150	0.12	19	-	-	-	-	0.32	45	449	0.75	13	8.7
175	-	-	-	-	-	-	0.28	39	469	0.78	26	16.6
200	-	-	-	-	-	-	0.29	41	330	0.55	6	5.5
Positive control	0.08	13	202	0.40	341	422.0	0.20	28	314	0.52	331	316.2

CE= total cloning efficiency

RCE= relative cloning efficiency

Conclusions:

Under the conditions of this study the test material did not cause a positive response in the non-activated and S9-activated systems and was concluded to be negative in this mutation assay.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guideline EPA 84-2, under GLP conditions.

The test material was tested in the CHO/HGPRT Mutation Assay in the absence and presence of Aroclor-induced rat liver S9.

The preliminary toxicity assay was used to establish the dose range for the mutagenesis assay. The mutagenesis assays was used to evaluate the mutagenic potential of the test material. Sterile distilled water was chosen by the Sponsor as the solvent for the test material. The test material was soluble in sterile distilled water at a concentration of 14.3 mg/mL, the maximum concentration prepared.

In the preliminary toxicity assay, the maximum concentration tested was 1430 μg/mL (10 mM). There was no visible precipitate in the treatment medium at any concentration. Selection of dose levels for the mutagenesis assay was based on the cloning efficiency relative to the solvent control. Substantial toxicity, i.e., cloning efficiency < 50 % of the solvent control, was observed at≥505 μg/mL with and without S9 activation. Based on these findings, the doses chosen for the mutagenesis assay ranged from 100 to 600 μg/mL for the nonactivated cultures and 100 to 500 μg/mL for the S9-activated cultures.

In the mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies > 40 mutants per 10^6 clonable cells, were observed. No visible precipitate was observed in treatment medium at any concentration. Toxicity, i.e., cloning efficiency≤50 % of the solvent control, was observed at doses of≥125 μg/mL without activation and≥150 μg/mL with activation. 

In the presence of S9 RCE reduced to 41%, not considered sufficient in accordance with the guideline requirements (10-20%) RCE reduced to 25%. Whilst not considered acceptable with guidance (cytotoxicity 10-20%), in the absence of a dose related increase in mutant frequency, a reduction in RCE to 25% was deemed acceptable

However, in the absence of S9 mutant frequency was not determined due to contamination in the mutation plates. Instead a dose of 125 mg/mL (the next highest concentrations) was assessed for mutant frequency, where RCE was reduced to 25 %. In the absence of any dose related increase in mutant frequency up RCE of 25 % this level of toxicity was deemed acceptable. In the presence of S9 RCE was reduced to 41 % at a concentration of 200 µg/mL where mutant frequency was determined.

Under the conditions of this study, the test material was concluded to be negative in the CHO/HGPRT Mutation Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

IN VIVO STUDIES

Mouse Micronucleus Test

The genetic toxicity of the test material was investigated in accordance with the standardised guideline 84-2, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested in the mouse micronucleus assay. The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot toxicity study followed by a toxicity study. The second phase, the micronucleus study, evaluated the potential of the test material to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. In both phases of the study, test and control materials were administered in a constant volume of 20 mL/kg body weight by a single intraperitoneal injection.

Water was determined to be the solvent of choice based on a request by the Sponsor and compatibility of the vehicle with the test system animals. The test material was soluble in water at 14 mg/mL, the maximum concentration tested. Dosing concentrations were delivered to the test system as clear, colourless solutions.

In the pilot toxicity study, male mice were dosed with 100, 120, 180 or 225 mg/kg body weight and male and female mice were dosed with 280 mg/kg. No mortality was observed in any male or female test material treated group. Clinical signs, observed three days after dose administrations, included: lethargy in males at 180 and 225 mg/kg and lethargy and piloerection in males and females at 280 mg/kg . In this phase of the study, the maximum tolerated dose was not established and the toxicity study was conducted. In the toxicity study, the test material dosing solutions were prepared following the Sponsor's instructions. Male and female mice were dosed with 50, 100, 200 or 280 mg/kg body weight. Mortality was observed in 3/5 male mice and 4/5 female mice at 200 mg/kg and in 4/5 males and 4/5 females at 280 mg/kg. Clinical signs following dose administration included: lethargy and piloerection in males and females at 200 and 280 mg/kg, and tremors in males and females at 280 mg/kg. In addition, crusty eyes were noted in males at 200 and 280 mg/kg and crusty nose in females at 280 mg/kg. The high dose for the micronucleus test was set at mg/kg which was estimated to be the maximum tolerated dose.

In the micronucleus assay, male and female mice were dosed with 25, 50 or 100 mg/kg body weight. No mortality or clinical signs were observed in any male or female mice in the micronucleus study. All animals treated with the test or control materials appeared normal following dose administration. Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Reductions (up to 20 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test material-treated groups relative to the respective vehicle controls. These reductions suggest that the test material did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test material-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration. The positive control, cyclophosphamide, induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice, all criteria for a valid test were met.

Under the conditions of this study the test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.

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:

16 January 2001 and 4 March 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: Toxicity study: males: 27.7 to 35.1 g and female: 24.6 to 31.0 g and Micronucleus assay: males: 28.4 to 33.7 g and female: 25.8 to 29.3 g.
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and which were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 °F
- Humidity: 50 ± 20 %
- Photoperiod: 12 hour light/dark cycle

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: sterile distilled water
- Justification for choice of solvent/vehicle: Water was determined to be the solvent of choice based on a request by the Sponsor and compatibility of the vehicle with the test system animals. The test material was soluble in water at 14 mg/mL, the maximum concentration tested in the study.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dosing solutions were prepared in an appropriate sized, sealed amber glass vials.
- Half of the needed amount of solvent was added and the vials were sealed. In each vial, the appropriate amount of the test material was injected through the sealed stopper. To achieve the proper concentration of dosing solutions, the amount of additional solvent was adjusted according to the measured amount of the test material.
- Dose administration was conducted within, approximately, 30 minutes of preparation.

Duration of treatment / exposure:

Single administration

Frequency of treatment:

Single treatment

Post exposure period:

Bone marrow harvested at 24 and 48 hrs post dosng

Dose / conc.:

25 mg/kg bw (total dose)

Dose / conc.:

50 mg/kg bw (total dose)

Dose / conc.:

100 mg/kg bw (total dose)

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: intraperitoneal injection
- Doses / concentrations: 50 mg/kg
- Cyclophosphamide was dissolved in sterile distilled water at a concentration of 2.5 mg/mL.

Tissues and cell types examined:

Bone marrow; 2000 polychromatic erythrocytes (PCEs) examined/animal for the presence of micronuclei. For toxicity assessment, the proportion of PCE to total erythrocytes was recorded (number of PCE in 1000 erythrocytes scored).

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- Toxicity Study doses: 50, 100, 200 or 280 mg/kg body weight.
- Based on the results of the toxicity study the high dose for the micronucleus test was set at 100 mg/kg for male and female mice which was estimated to be the maximum tolerated dose.
- Micronucleus Assay doses: 25, 50, 100 mg/kg body weight. An additional group of five males and five females were designated as replacement animals in the event of mortality prior to the scheduled sacrifice time and was dosed with the test material high dose level.

TREATMENT AND SAMPLING TIMES:
- Bone Marrow Collection was performed at 24 and 48 hours after dose administration.
- Vehicle: 5 animals at 24 hours and 5 animals at 48 hours
- 25 mg/kg test material: 5 animals at 24 hours
- 50 mg/kg test material: 5 animals at 24 hours
- 100 mg/kg test material: 5 animals at 24 hours and 5 animals at 48 hours
- Positive control: 5 animals at 24 hours


DETAILS OF SLIDE PREPARATION:
- At the scheduled sacrifice times, five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum.
- The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL foetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet.
- The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
- Slides were coded using a random number table by an individual not involved with the scoring process.
- Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte. The number of micronucleated normochrornatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Evaluation criteria:

EVALUATION OF RESULTS
- The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group.
- In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
- All conclusions were based on sound scientific judgement; however, as a guide to interpretation of the data, the test material was considered to induce a positive response if a dose responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p ≤ 0.05, Kastenbaum-Bowman Tables) at any sampling time.
- If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended.
- The test material was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

CRITERIA FOR A VALID TEST
- The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5 %) in the vehicle control.
- The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p ≤ 0.05, Kastenbaum-Bowman Tables).

Statistics:

The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TOXICITY ASSAY
- Mortality occurred after dose administration as follows: 3/5 male mice and 4/5 female mice at 200 mg/kg and 4/5 males and 4/5 females at 280 mg/kg.
- Clinical signs, which were noted on the days following dose administration included: lethargy and piloerection in all males and all females at 200 mg/kg, but only in 3/5 males and 4/5 females at 280 mg/kg. Tremors were observed in 2/5 males and 1/5 females at 280 mg/kg. In addition, crusty eyes and/or a crusty nose were observed in some mice at 200 and 280 mg/kg. All other mice appeared normal throughout the observation period.
- A loss in animal body weight from approximately 3 to 18 % was recorded in all of the test material treated groups. The high dose for the micronucleus test was set at 100 mg/kg for male and female mice which was estimated to be the maximum tolerated dose.

MICRONUCLEUS ASSAY
- No mortality occurred at any dose level during the course of the micronucleus study.
- All mice treated with test or control materials appeared normal throughout the observation period.
- The incidence of micronucleated polychromatic erythrocytes per 10000 polychromatic erythrocytes were scored (2000 PCEs/animal). Reductions of 2 to 20 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test material-treated groups relative to their respective vehicle controls. These reductions suggest that the test material did not inhibit erythropoiesis. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test material-treated groups was not statistically increased relative to their respective vehicle control in either male or female mice, regardless of dose level or bone marrow collection time (p>0.05, Kastenbaum-Bowman Tables).
- The positive control, cyclophosphamide, induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p ≤ 0.05, Kastenbaum-Bowman Tables). All criteria for a valid test were met.

Table 1: Summary of Micronucleus test

Treatment (mg/kg)	Sex	Time (hours)	PCE/Total erythrocytes (Mean ± SD)	Change from control (%)	Micronucleated Polychromatic Erythrocytes
					Number per 1000 PCEs (Mean ± SD)	Number per PCE’s scored
Vehicle	M	24	0.451 ± 0.11	-	0.3 ± 0.27	3/ 1000
	F	24	0.470 ± 0.10	-	0.3 ± 0.27	3/ 1000
25	M	24	0.518 ± 0.05	15	0.3 ± 0.27	3/ 1000
	F	24	0.484 ± 0.04	3	0.3 ± 0.27	3/ 1000
50	M	24	0.443 ± 0.06	-2	0.4 ± 0.22	4/ 1000
	F	24	0.406 ± 0.05	-14	0.4 ± 0.22	4/ 1000
100	M	24	0.384 ± 0.09	-15	0.0 ± 0.00	0/ 1000
	F	24	0.374 ± 0.07	-20	0.5 ± 0.35	5/ 1000
Cyclophosphamide 50	M	24	0.323 ± 0.04	-28	24.7 ± 3.68	247/ 1000*
	F	24	0.313 ± 0.03	-33	23.2 ± 5.66	232/ 1000*
Vehicle	M	48	0.491 ± 0.05	-	0.1 ± 0.22	1/ 1000
	F	48	0.501 ± 0.05	-	0.4 ± 0.22	4/ 1000
100	M	48	0.425 ± 0.02	-13	0.5 ± 0.35	5/ 1000
	F	48	0.441 ± 0.02	-12	0.3 ± 0.27	3/ 1000

*= p≤0.05 (Kastenbaum-Bowman Tables)

Conclusions:

Under the conditions of this study the test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice .

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guideline 84-2, under GLP conditions.

The test material was tested in the mouse micronucleus assay. The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot toxicity study followed by a toxicity study. The second phase, the micronucleus study, evaluated the potential of the test material to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. In both phases of the study, test and control materials were administered in a constant volume of 20 mL/kg body weight by a single intraperitoneal injection.

Water was determined to be the solvent of choice based on a request by the Sponsor and compatibility of the vehicle with the test system animals. The test material was soluble in water at 14 mg/mL, the maximum concentration tested. Dosing concentrations were delivered to the test system as clear, colourless solutions.

In the pilot toxicity study, male mice were dosed with 100, 120, 180 or 225 mg/kg body weight and male and female mice were dosed with 280 mg/kg. No mortality was observed in any male or female test material treated group. Clinical signs, observed three days after dose administrations, included: lethargy in males at 180 and 225 mg/kg and lethargy and piloerection in males and females at 280 mg/kg . In this phase of the study, the maximum tolerated dose was not established and the toxicity study was conducted. In the toxicity study, the test material dosing solutions were prepared following the Sponsor's instructions. Male and female mice were dosed with 50, 100, 200 or 280 mg/kg body weight. Mortality was observed in 3/5 male mice and 4/5 female mice at 200 mg/kg and in 4/5 males and 4/5 females at 280 mg/kg. Clinical signs following dose administration included: lethargy and piloerection in males and females at 200 and 280 mg/kg, and tremors in males and females at 280 mg/kg. In addition, crusty eyes were noted in males at 200 and 280 mg/kg and crusty nose in females at 280 mg/kg. The high dose for the micronucleus test was set at mg/kg which was estimated to be the maximum tolerated dose.

In the micronucleus assay, male and female mice were dosed with 25, 50 or 100 mg/kg body weight. No mortality or clinical signs were observed in any male or female mice in the micronucleus study. All animals treated with the test or control materials appeared normal following dose administration. Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Reductions (up to 20 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test material-treated groups relative to the respective vehicle controls. These reductions suggest that the test material did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test material-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration. The positive control, cyclophosphamide, induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice, all criteria for a valid test were met.

Under the conditions of this study the test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Iodomethane is not genotoxic, and therefore classification is not required.