Reaction products of 1,4-cyclohexanedimethanol with propylene oxide and ammonia

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay

In a K1 bacterial reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535,TA1537 and E. coli WP2 uvrA, performed according to OECD Guideline 471 (Ames), it was concluded that the test substance has no mutagenic properties towards any of the bacterial strains tested in the absence and in the presence of S9-mix under the test conditions described in the report.

In Vitro Mammalian Cell Gene Mutation Test:

In a K1 mammalian cell gene mutation assay performed on Chinese Hamster Ovary according to the OECD guideline 476, it was concluded that the test substance is not mutagenic in the Chinese Hamster Ovary test system under the experimental conditions described in the report.

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:

2010-04-22 to 2010-05-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Jeffamine RFD-270 Amine
- Substance type: Colourless liquid
- Physical state: Liquid
- Lot/batch No.: 8802-8-9
- Analytical purity: 92%
- Composition of test material, percentage of components: 0.02 wt% water
- Purity test date: 2010-07-15
- Storage condition of test material: Sample stored in cool, well-ventilated storage area prior to testing

Target gene:

histidine (Salmonella strains) and tryptophan (E.coli strain)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1 (initial toxicity - mutation assay): 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (confirmatory mutagenicity assay): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: A solubility test was conducted using water to determine the highest soluble or workable stock concentration, up to 50 mg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 ; TA98, TA1535 and TA1537 = 1.0 µg/plate ; TA100 = 2.0 µg/plate ; WP2 uvrA = 10 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 ; TA98 = 1.0 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 ; TA100, TA1535 = 1.0 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 ; TA1537 = 75 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 ; WP2 uvrA = 1000 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation method

DURATION:
- Exposure duration: 48 to 72 hours

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate.
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.

NUMBER OF REPLICATIONS:
Initial toxicity-mutation assay: vehicle control, positive controls and eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.
Confirmatory mutagenicity assay: All dose levels of test article (minimum five), vehicle control and positive controls were plated in triplicate.

NUMBER OF CELLS EVALUATED: Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 0.3E09 cells per milliliter.

DETERMINATION OF CYTOTOXICITY: reduction in mean number of revertants

Evaluation criteria:

For the test article 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 article.
Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-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 was greater than or equal to 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

(no precipitate and no toxicity were observed)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity

Remarks:

(no precipitate and no toxicity were observed)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: During incubation of the assay plates from the initial toxicity-mutation assay, a technical error in deviation from the protocol was discovered in which the assay plates from the initial assay did not incubate for a full 48-hour period. The entire initial toxicity-mutation was not evaluated but was retested. In the retest of the initial toxicity-mutation assay, the maximum dose tested was 5000 µg/plate; this dose level was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. No positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation or with tester strains TA98, TA100, TA1535 and WP2 uvrA in the absence of S9. No precipitate was observed. Toxicity was observed at 5000 µg per plate with tester strain TA100 in the absence of S9 activation only. Due to an unacceptable vehicle control value, tester strain TA1537 in the absence of S9 activation was not evaluated for mutagenicity but was retested based on the precipitate and toxicity profiles observed. Based on the findings of the retest of the initial toxicity-mutation assay, the maximum dose plated in the retest and confirmatory mutagenicity assays was 5000 µg/plate.
- Water solubility: The test article was considered to be soluble in water.
- Sterility results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions and the S9 and Sham mixes.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants per plate for the negative and positive control substances were in the range of the historical control values of the laboratory.

In Experiment B2 an unacceptable vehicle control value has been observed and tester strain TA1537 in the absence of S9 activation was not evaluated for mutagenicity but was retested in Experiment B3 based on the precipitate and toxicity profiles observed. Based on the findings of the retest of the initial toxicity-mutation assay, the maximum dose plated in the second retest and confirmatory mutagenicity assays was 5000 μg per plate. In Experiment B3 (Second Retest of the Initial Toxicity-Mutation Assay), no positive mutagenic response was observed with tester strain TA1537 in the absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor appreciable toxicity was observed. A reduction in revertants was observed at 5000 μg per plate with tester strain TA1535 in the presence of S9 activation.

Confirmatory mutagenicity assay:

The dose levels tested were 50, 150, 500, 1500 and 5000 µg per plate. Neither precipitate nor background lawn toxicity was observed. In this experiment no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Conclusions:

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the substance did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-12-15 to 2012-01-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: F12FBS5-Hx
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: Cells used in the mutation assay were within four subpassages from cleansing in order to assure karyotypic stability
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity assay: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 2100 µg/mL
Mutagenesis assay: 500, 1000, 1500, 1750 and 2100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distlled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

used at stock concentration of 20 µL/mL, for a final concentration of 0.2 µL/mL as positive control for non-activated test system

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

used at a stock concentration of 400 µg/mL, for a final concentration of 4 µg/mL, as positive control for the S9-activated test system

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 7 to 9 dyas
- Selection time (if incubation with a selection agent): 7 to 10 days

SELECTION AGENT (mutation assays): After the expression period, cells are grown in medium with and without 6-thioguanine.

NUMBER OF REPLICATIONS: duplicates, both in the absence and the presence of S9 activation. For cloning efficiency determinations at the time of selection, 100 cells/60 mm dish were plated in triplicate in F12FBS5+Hx without TG.

NUMBER OF CELLS EVALUATED: 2x1E06 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

- The test article was considered to induce a positive response if there was a concentration-related increase in mutant frequencies with at least two consecutive concentrations showing mutant frequencies > 40 mutants per 1E06 clonable cells.
- If a single point above 40 mutants per 1E06 clonable cells was observed at the highest concentration, the test article was considered suspect
- If no culture exhibited a mutant frequency of > 40 mutants per 1E06 clonable cells, the test article was considered negative.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 1E06 clonable cells

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity, i.e. relative cloning efficiency < 50%, was observed only at 2100 µg/mL without S9 activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: The osmolality of the solvent control was 319 mmol/kg and the osmolality of the top concentration, 2100 μg/mL, was 287 mmol/kg.
- Evaporation from medium: no data
- Water solubility: The substance was soluble in water at a concentration of 50 mg/mL, the maximum concentration tested in the solubility test. The test article was found to be workable in water at >=5.0 mg/mL and soluble at <= 1.5 mg/mL in the preliminary toxicity assay.
- Precipitation: No test article precipitate was observed at any concentration in treatment medium.

RANGE-FINDING/SCREENING STUDIES: Based on the results of the preliminary toxicity test, the concentrations chosen for the mutagenesis assay ranged from 500 to 2100 μg/mL for both the non-activated and S9-activated cultures. There was no visible precipitate in the treatment medium at any concentration. Selection of concentrations 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 (relative cloning efficiency), was observed only at 2100 µg/mL without S9 activation.

Conclusions:

Based on the results of the CHO/HGPRT Mutation Assay, under the conditions of this study, (i.e. no dosing formulation analysis and no stability information about the neat test article), the test substance was concluded to be negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In a K1 Mouse Bone Marrow Erythrocyte Micronucleus Test performed according to the guideline OECD 474, the test substance was considered to be negative under the experimental conditions described in the report.

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:

2011-12-19 - 2012-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

other: ICH S3A (1995), ICH S2A (1996), ICH S2B (1997)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Jeffamine RFD-270 Amine
- Substance type: Colourless liquid
- Physical state: Liquid
- Lot/batch No.: 8802-8-9
- Analytical purity: 92%
- Composition of test material, percentage of components: 0.02 wt% water
- Purity test date: 2010-07-15
- Storage condition of test material: Sample stored in cool, well-ventilated storage area prior to testing

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan
- Age at study initiation: 6 weeks old
- Weight at study initiation: dose range finding study: male mice: 30.9 - 36.5 grams, female mice: 30.0 - 25.0 grams; definitive micronucleus study: male mice: 29.5 - 34.0 grams, female mice: 24.4 - 28.6 grams
- Assigned to test groups randomly: yes, based on equalization of group mean body weights. At the time of randomization, the weight variation of animals did not exceed ± 20% from their group mean.
- Fasting period before study: No
- Housing: Animals were housed by sex, with up to five animals per rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-Vent full ventilation, HEPA filtered system.
- Diet (e.g. ad libitum): Certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet); ad libitum
- Water (e.g. ad libitum): Tap water; Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ± 22.2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: All test substance dose formulations were freshly prepared prior to administration. An appropriate amount of the test substance was weighed separately for each formulation, and ~ 80% of the required vehicle was added to the respective containers. Each formulation was vortexed briefly, and the remaining volume of the vehicle was added to achieve the required final volume. All formulations appeared clear formulations.

Duration of treatment / exposure:

Animals were treated once.

Frequency of treatment:

Animals were treated once, by oral gavage, using a dose volume of 20 mL/kg

Post exposure period:

Bone marrow was harvested at 24 or 48 hours after treatment and evaluated microscopically for the presence of micronucleated polychromatic erythrocytes (MPCEs), as well as for the proportion of PCEs (relative to total erythrocytes) as an indication of bone marrow cytotoxicity.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

125 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5, with exception of the high dose group (500 mg/kg) where 15 mice/sex were tested (including 5 replacement mice/sex)

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg

Tissues and cell types examined:

bone marrow ; polychromatic erythrocytes were examined for the presence of micronuclei

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: 24 hours post dose: 5 animals per group, 48 hours post dose: 5 animals in the vehicle control group and 5 animals in the high dose group

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): sampling at 24 and 48 hours post dose

DETAILS OF SLIDE PREPARATION: The bone marrow was transferred to a labeled centrifuge tube containing approximately 1 mL fetal bovine serum, the cells were pelleted by centrifugation at approximately 100 x g for five minutes, and the supernatant was removed, leaving a small amount of serum with the remaining pellet.

The cells were re-suspended and a small drop of the suspension was spread onto a clean glass slide. Two slides were prepared from each mouse, labeled with the experiment and animal numbers, air dried, and fixed in methanol. One set of slides was stained with acridine orange and anallyzed microscopically.

METHOD OF ANALYSIS: Slides were initially scanned using a fluorescent microscope and medium magnification (400X; blue 440-490 nm excitation filter and 520 nm barrier filter) and an area acceptable quality was selected such that the cells were well spread and stained. Once selected, the following cell populations and cellular components were evaluated under high power magnification (1000X oil immersion).

- Polychromatic erythrocytes (PCEs):
PCEs are young erythrocytes in the early stage of erythropoiesis that stain orange-red; 2000 PCEs/animal (10000 PCEs/group) were scored for the presence of micronuclei.
- Normochromatic erychtrocytes (NCEs):
NCEs are mature erythrocytes, the final cell population formed during erythropoiesis, that stain light green; the number of NCEs/1000 total erythrocytes also was determined for each animal, to calculate the proportion of PCEs to total erythrocytes, as an index of bone marrow cytotoxicity. PCE proportions less than 20% of control values are considered excessively toxic.
- Micronuclei (M)
Micronuclei are round nuclear (chromosome) fragments, generally with a sharp contour and diameters of approximately 1/20 to 1/5 of an erythrocyte, that stain green. Micronuclei may occur in PCEs (MPCEs) or NCEs (MNCEs). The frequency of MNCEs observed while scoring 2000 PCEs was determined, but the results are not presented or used in analysis of genotoxic response since the primary target cells are the PCEs.

Evaluation criteria:

- A test substance is considered to be positive for the induction of micronuclei if it induces a significant increase in MPCE frequency (p<=0.05) at any dose level of sampling time
- A test substance is considered to be negative for the induction of micronuclei if no significant increase in MNPCE frequency is observed (p>0.05).
- If criteria for either a positive or negative clastogenic response were not met, the resuls would have been judged as equivocal.
Other criteria also may be used in reaching a conclusion about the study results(e.g. magnitude of any increase, dose-dependency, comparison to historical control values, biological significance, etc.).

Statistics:

Statisical significance, as compared to the concurrent vehicle control, was determined for each group using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately by sex and sampling time.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Complete mortality was observed on day 0 in all mice treated with Jeffamine RFD-270 at doses of 750, 1000 and 2000 mg/kg. All mice treated at a dose of 500 mg/kg exhibited piloerection after treatment and on day 1; males in this group also exhibited lethargy on day 0. All mice in the 100 and 250 mg/kg treatment groups appeared normal throughout the study, except for piloerection noted in the 100 mg/kg dose group on day 3.

RESULTS OF DEFINITIVE STUDY
Five males treated with Jeffamine RFD-270 at a dose of 500 mg/kg were found dead on day 0. Males from the replacement group were reallocated to ensure sufficient animals were available for each sampling time. Piloerection also was observed in some or all males at all dose levels on day 0 and 1, and in some or all females at a dose of 500 mg/kg on day 0 and 1. Lethargy also was observed in all males at a dose of 500 mg/kg on day 0.

Conclusions:

The results of this test indicate the test substance was negative in the mouse bone marrow erythrocyte micronucleus.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

An Ames-test was performed according to a method equivalent to OECD Guideline 471 and in compliance with the principles of GLP with the following Salmonella typhimurium strains: TA 1535, TA 1537, TA 98, TA 100 and Escherichia Coli strainWP2 uvrA (Wagner and VanDyke, 2011). The test substance was tested with and without metabolic activation system, according to the plate incorporation method. The test article formed a soluble and clear solution in water at 50 mg/mL.

In the initial toxicity mutation assay, the dose level tested were 1.5, 5.0, 15, 50, 500, 1500 and 5000 µg/plate. In this first assay, no positive mutagenic responses were observed with any of the tester strains in the presence or absence of metabolic activation. No precipitate was observed. Toxicity was observed at 5000 μg per plate with tester strain TA100 in the absence of S9 activation only. Due to an unacceptable vehicle control value, tester strain TA1537 in the absence of S9 activation was not evaluated for mutagenicity but was retested based on the precipitate and toxicity profiles observed. Based on the findings of the retest of the initial toxicity-mutation assay, the maximum dose plated in the retest and confirmatory mutagenicity assays was 5000 μg per plate.

In the second retest of the initial toxicity-mutation assay, no positive mutagenic response was observed with tester strain TA1537 in the absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor appreciable toxicity was observed. In the confirmatory mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed. A reduction in revertants was observed at 5000 μg per plate with tester strain TA1535 in the presence of S9 activation.

Under the conditions of this study, the substance was concluded to be negative in the Bacterial Reverse Mutation Assay.

The substance was tested in the CHO/HGPRT mutation assay in the absence and presence of a metabolic activation system. The preliminary toxicity assay was used to establish the concentration range for the initial mutagenesis assay. The mutagenesis assay was used to evaluate the mutagenic potential of the test article.

Sterile distilled water was determined to be the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in water at a concentration of 50 mg/mL, the maximum concentration tested in the solubility determination. The test article was found to be workable in water at ≥5.0 mg/mL and soluble at ≤1.5 mg/mL in the preliminary toxicity assay. In the preliminary toxicity assay, the maximum concentration of the substance tested was 2100 μg/mL (10 mM). There was no visible precipitate in the treatment medium at any concentration. Selection of concentrations 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 (relative cloning efficiency), was observed only at 2100 μg/mL without S9 activation. Based on these findings, the concentrations chosen for the mutagenesis assay ranged from 500 to 2100 μg/mL for both the non-activated and S9-activated cultures.

In the mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies >40 mutants per 1E06 clonable cells, were observed. No visible precipitate was observed in treatment medium at any concentration. Toxicity, i.e., relative coning efficiency <50%, was observed only at 2100 μg/mL without S9 activation.

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

Genetic toxicity in vivo:

The test article was evaluated in the Mouse Bone Marrow Erythrocyte Micronucleus Test for its ability to induce micronuclei in the polychromatic erythrocytes (PCE) in the bone marrow of male and female ICR mice.

Based on the mortality and clinical signs observed in a preliminary dose range finding study, the test substance was evaluated in the definitive micronucleus test at doses of 125, 250 and 500 mg/kg. Cyclophosphamide and purified water were evaluated concurrently as the positive and vehicle controls, respectively. All animals were treated once, by oral gavage, using a dose volume of 20 mL/kg. Mice were observed after dose administration and at least daily throughout the course of the study for clinical signs of toxicity. Bone marrow was harvested at 24 or 48 hours after treatment and evaluated microscopically for the presence of micronucleated PCEs (MPCEs), as well as for the proportion of PCEs (relative to total erythrocytes) as an indication of bone marrow

cytotoxicity.

Five males treated with the test substance at a dose of 500 mg/kg were found dead on Day 0. Surviving males in the replacement group were reallocated to ensure sufficient animals were available for each sampling time. Piloerection also was observed in some or all males at all dose levels on Day 0 and 1, and in some or all females at a dose of 500 mg/kg on Day 0 and 1. Lethargy also was observed in all males at a dose of 500 mg/kg on Day 0.

No significant decreases in the proportion of PCEs, or significant increases in the frequency of MPCEs, were observed for the test substance at any dose level or sampling time (p> 0.05). In contrast, the cyclophosphamide positive control induced a significant increase in MPCE frequencies (p< 0.05).

All positive and vehicle control values were within accceptable ranges, and all criteria for a valid study were met.

These results indicate that the substance was negative in the Mouse Bone Marrow Erythrocyte Micronucleus Test under the conditions, and according to the criteria of the test protocol.

Justification for classification or non-classification

Based on the available data on the substance and according to the criteria of the CLP Regulation, the test substance should not be classified as a mutagenic substance.