2-[(2-[2-(dimethylamino)ethoxy]ethyl)methylamino]ethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse Mutation Assay (OECD guideline 471):

Two reliable studies are available with the test substance (Barfknecht et al., 1984; confirmation assay Pharmakon research International, Inc., 1990). The substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium in the presence and absence of metabolic activation.

Chromosome aberration assay in lymphocytes (equivalent to OECD 473):

The test substance showed some evidence of clastogenic activity at the 15 -hour harvest (Adams et al., 1992). However, results generated from 10 -hour harvest and 24 -hour harvest were clearly negative. The overall conclusion was that the test substance provided an equivocal response in the assay.

Gene mutation in mammalian cells (equivalent to OECD 476):

The structurally analogue source substance was not active in producing gene mutations in CHO cells (BRRC, 1983). The same is assumed for the test substance.

In vitro transformation assay:

The test substance did not induce the appearance of a significant number of transformed foci over the concentration range of 20 to 188 nl/ml (Litton Bionetics Inc., 1983). Therefore, the test substance was considered to be inactive in the assay.

DNA damage and repair (equivalent to OECD 482):

The test substance was determined to be negative based upon its inability to produce a mean net nuclear grain count greater than or equal to 5 times the vehicle control at concentrations up to cytotoxic levels (Pharmakon Research International, Inc., 1985)

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:

1984-02-12 to 1984-02-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No analytical provided on test substance, an E. coli strain was not included in study, and negative result was not confirmed in a repeat assay and no justification for not repeating assay was provided. However the study was GLP-compliant, positive and vehicle control were included and the study was performed in the presence and absence of metabolic activation.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No analytical provided on test substance, an E. coli strain was not included in study, and negative result was not confirmed in a repeat assay and no justification for not repeating assay was provided.

GLP compliance:

yes (incl. QA statement)

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): 5601-22-20, Order #J-203
- Analytical purity: purity was the responsibility of the sponsor
- Physical state: yellow liquid
- Storage condition of test material: the test substance was stored at room temperature in a clear glass bottle

Target gene:

histidine locus

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

100, 333, 1000, 3333 and 10,000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: test substance was determined to be soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation Migrated to IUCLID6: used for TA1535 and TA100 at 10 ug/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation Migrated to IUCLID6: used for TA1537 at 150 ug/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation Migrated to IUCLID6: used for TA1538 and TA98 at 5 ug/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene used for all strains at 5 ug/plate

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 hours
- Selection time (if incubation with a selection agent): 48-72 hours (simultaneous with exposure)

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 3 (test substance), 2 (positive and vehicle control)

DETERMINATION OF CYTOTOXICITY
- Method: reduction in bacterial background lawn

Evaluation criteria:

A positive result was defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modifications of the original doses in a repeat assay. If the solvent control was within one standard deviation of the historical mean for control values and the test substance produced the highest increase equal to or greater than three times the solvent control value, the test substance was considered positive. A negative result was defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

No data

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

but no toxicity was observed up to 10000 ug/plate in the range-finding assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity assay was performed with TA1538 and TA100 in the presence and absence of metabolic activation. Tester strains were treated with the test substance at dose levels of 100-10,000 µg/plate for 48 hours using the plate incorporation assay. Following exposure, background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria. No toxicity was observed up to 10000 ug/plate in the presence or absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
All solvent and positive control values were within the acceptable limits of mean historical data.

all strains/cell types tested

Conclusions:

The test substance was evaluated for induction of mutagenicity in the Ames Salmonella/Microsome Plate test in the presence and absence of metabolic activation using S. typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA1538. The test substance failed to induce reverse mutations in any of the tester strains both in the presence and absence of metabolic activation up to concentration levels of 10,000 ug/plate.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-04-06 to 1990-04-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No analytical provided on test substance, an E. coli strain was not included in study, and negative result was not confirmed in a repeat assay and no justification for not repeating assay was provided. However study was GLP, positive and vehicle control were included and the study was performed in the presence and absence of metabolic activation.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No analytical provided on test substance, strain TA102 or an E. coli strain was not included in study, and negative result was not confirmed in a repeat assay and no justification for not repeating assay was provided.

GLP compliance:

yes (incl. QA statement)

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): 6398-16-20
- Physical state: Clear, pale yellow liquid
- Storage condition of test material: room temperature in clear glass container

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA1535, TA1537, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

167, 500, 1670, 5000, 7500 and 10000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO, lot #887134, supplied by Fisher Scientific (Fairlawn, NJ)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

see below

Remarks:

with and without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without activation, used for TA1535 and TA100 at 10 ug/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without activation, used for strain TA1537 at 150 ug/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without activation, used for strains TA1538 and TA98 at 5 ug/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

no

Positive control substance:

other: 2-anthramine- used for all strains at 2.5 ug/plate

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: ~48 hours
- Selection time (if incubation with a selection agent): ~ 48 hours (simultaneous with exposure)

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: the background lawn and spontaneous revertants were scored for normal, inhibited or no growth. Inhibited growth was characterized by the absence of a confluent bacterial lawn and/or the presence of pindot colonies.

OTHER: to ensure the sterility of solvent, compound and equipment, standard contamination evaluations were performed wih the assay. The solvent, top agar, S9 mix, and top dose of the test substance were evaluated at the same volumes used in the assay. The test substance, solvent and S9 mix were evaluated as in the mutation assay, but in the absence of added tester strains. Top agar alone was also plated on minimal glucose plates. All plating was done in triplicate. Plates were incubated for 48 hours and then scored for bacterial growth.

Evaluation criteria:

A positive result was defined as a statistically significant, dose-dependent increase in the number of histidine-independent revertants with at least one dose level inducing a revertant frequency that was two-fold the spontaneous solvent control value. If the test substance did not induce a statistically significant, dose-dependent increase in revertant frequency but did induce a revertant frequency at one dose level that was two-fold the spontaneous control value, the result was considered equivocal. A negative result was defined as the absence of a statistically significant or dose-dependent increase in the number of histidine-independent revertants.

Statistics:

Statistical anlayses were performed using the program developed by Snee and Irr (1981), with significance established at the 95 % confidence limit.

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA1535, TA1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: the toxicity of the test substance was evaluated by treating duplicate cultures of strains TA1538 and TA100 for ~48 hours using the plate incorporation assay with five doses in the absence of metabolic activation. The background lawn and spontaneous revertants were scored for normal, inhibited or no growth. Inhibited growth was characterized by the absence of a confluent bacterial lawn and/or the presence of pindot colonies. Results of the toxicity study indicated that the test substance was not toxic to either strain at doses of 50, 167, 500, 1670 and 5000 ug/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: All positive and solvent control values were within acceptable limits.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Inhibited growth was observed in TA100 at doses of 5000 and 7500 ug/plate with S9. Therefore, the test substance was re-evaluated in TA100 and the same dose levels in the presence of S9. Normal growth was observed in the repeat assay at all dose levels, and therefore the toxicity in the original assay was considered to be a spurious result due to technical error.

all strains/cell types tested

Conclusions:

The test substance was evaluated for mutagenic potential using the Ames/Salmonella Plate Incorporation Assay in Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA1538 in the absence and presence of metabolic activation. The test substance was negative in all tester strain both in the presence and absence of metabolic activation under the conditions of the study up to a concentration of 10000 µg/plate.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-02-20 to 1992-04-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study performed similar to Guideline and under GLP, however it was only conducted with metabolic activation. 3 dose levels were used for analysis, two harvest times were included, and positive and negative controls were included.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

The study was not conducted in the absence of metabolic activation.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): Texacat ZF-10; 2-(2-(2-dimethylaminoethoxy)-ethyl-methyl-amino)-ethanol
- Physical state: clear, colorless liquid
- Analytical purity: 98.3% GLC
- Purity test date: 1991-10-31
- Storage condition of test material: room temperature in the dark

Species / strain / cell type:

lymphocytes: obtained from healthy human male donors

Metabolic activation:

with

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

9.8, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/ml (initial assay with a 10 or 15 hour harvest)
625, 1000, 1250, 1500, 1750 adn 2000 µg/ml (repeat assay with a 15 hour harvest)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: Solvent was chosen based on solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: 20 and 25 ug/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 5 or 10 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Cells were harvested 10 or 15 hours after initiaiton of treatment. Colchicine was added during the last 2 hours before harvest.

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): Giemsa (dilution factor: 1 part Giemsa to 9 parts buffered distilled water)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphases per replicate where possible (only cells with 44-46 chromosomes were analyzed)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER: Prior to exposure, cells were incubated in the presence of phytohaemagglutinin for 48 hours to stimulate division.

Statistics:

The number of aberrant metaphase figures in each treatment group was compared with the solvent control value using Fisher's test.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the medium was measured at the higher concentrations of the test substance. The pH at all the concentrations analyzed was within the acceptable range for the test system.

COMPARISON WITH HISTORICAL CONTROL DATA: see below

ADDITIONAL INFORMATION ON CYTOTOXICITY: in the presence of metabolic activation, 1250 ug/ml of the test substance caused a reduction in mitotic index of 49 % of the solvent control value when harvested at 10 hours and 71 % at the initial 15 hour harvest. Concentrations selected for further analysis for both harvest times were selected as 1250, 625 and 156 ug/ml based on these results.
In the repeat of the 15 hour harvest, a dose related decrease in mitotic index was seen. The highest concentration selected for analysis, 1250 ug/ml, reduced the mitotic index to 28 % of the solvent control value. Other doses chosen for analysis were 1000 and 625 ug/ml.
The concentration of the positive control used in the initial assay at both harvest timepoints was 25 ug/ml because it gave a better quality of metaphase figures for analysis. The concentration of the positive control used for the repeat assay of the 15 hour harvest was 20 ug/ml because 25 ug/ml was too toxic.

When harvested 10 hours after the initiation of treatment, the test substance caused no statistically significant increases in the proportion of aberrant cells, at any concentration analysed. When harvested after 15 hours, statistically significant increases in the number of cells containing chromosome aberrations were seen at 625 ug/ml (p<0.05 excluding and including gaps) and 1250 ug/ml (p<0.01 excluding gaps and p<0.001 including gaps). However, all these increased lied within the historical control range (0 -5.25 % excluding gaps and 0 -6.25 % including gaps). In order to clarify the results obtained the test was repeated at the 15 hour harvest. In the repeat test, statistically significant (p<0.01) increases in the proportion of metaphase figures with chromosome damage were observed at 1000 and 1250 ug/ml. Due to the reproduciblity of the reponse seen at the 15 hour harvest, it was determined that the test substance had shown some evidence of clastogenic activity at this harvest timepoint. However, since the results generated from the 10 hour harvest in this study, and the 24 hour harvest from a previous study (data not provided) were clearly negative, the overall conclusion was that the test substance provided an equivocal response in the assay. The positive control substance caused large statistically significant increases in the proportion of aberrant cells. This demonstrated the sensitivity of the test system and the efficacy of the S9 mix.

Conclusions:

The test substance was evaluated for induction of chromosome aberrations in primary human lymphocytes treated in the presence of metabolic activation. The test substance showed some evidence of clastogenic activity at the 15 hour harvest. However, since the results generated from the 10 hour harvest in this study, and the 24 hour harvest from a previous study (no data provided) were clearly negative, the overall conclusion was that the test substance provided an equivocal response in the assay.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985-08-01 to 1985-09-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study performed similar to OECD guideline and per GLP. Only 60 cells evaluated per dose, but positive and negative controls were used, and adequate number of concentration levels were tested.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

yes

Remarks:

Brief analytical provided on test substance, and only a total of 60 cells per dose group were evaluated.

GLP compliance:

yes (incl. QA statement)

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Specific details on test material used for the study:

- Name of test material (as cited in study report): 5601-22-20
- Physical state: clear yellow liquid
- Analytical purity: 97 %
- Storage condition of test material: stored in glass bottle

Species / strain / cell type:

hepatocytes: obtained from male Fischer 344 rat

Metabolic activation:

without

Test concentrations with justification for top dose:

0.16, 0.5, 1.6, 5.0, 16, 50, 166, 500, 1666 and 5000 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle:suggestion of the sponsor

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

see below

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-acetamidofluorene at E-5 M

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18-20 hours (with 10uC/ml of 3H-thymidine present during exposure, specific activity 50-80 Ci/mM)
- Fixation time (start of exposure up to fixation or harvest of cells): After exposure, cells were washed three times with phosphate buffered saline. Cells on coverslips were swelled in 1 % sodium citrate for 10-15 minutes and fixed in three 30 minute changes of 100 % ethanol:glacial acetic acid (3:1). The coverslips were air dried and mounted cell surface up on glass slides with permount. Slides were dipped in NTB-2 photographic emulsion in the dark, allowed to dry overnight and stored at 4 degrees C in light proof boxes containing drierite as a desiccant. 7 days later, autoradiographs were developed in D19 for 4 minutes at 15 degrees C, washed in distilled water with glacial acetic acid for 30 seconds, immersed in Fixer for 10 minutes, washed in running tap water for 5 minutes, dried, stained in Harris Alum hematoxylin followed by a dip rinse in acid alcohol with rinsing in running tap water for 2-5 minutes and a dip rinse in ammonium water. Slides were then rinsed in running tap water for 2-5 minutes, dipped in 70 % ethyl alcohol, followed by a 10-60 second dip in eosin solution. The slides were then rinsed in 3 separate baths of 95 % ethyl alcohol for 2 minute intervals, followed by rinsing in three separate baths of 100 % ethyl alcohol for 2 minute intervals. The slides were air dried, and coverslipped in permount. Excess emulsion was scraped off.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: A total of 60 cells/dose point were counted for autoradiographic UDS determinations.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was evaluated by visual inspection (abnormal cell morphology). No further data was provided on the determination of cytoxicity.

Evaluation criteria:

The test substance was reported positive when the minimum net grain count of 5 per nuclei was consistently observed in triplicate wells. Where possible a dose response profile would have been observed. Due to the need to initially screen a substance over a wide range of doses, an adequate dose response may have not been obtained, and the test substance may have been classified as a "suspect" genotoxicant. To resolve the genotoxic potential of the substance, the sponsor may have chosen to initiate a second experiment with dose levels closely bracketing the positive response which resulted in classifying the substance as a "suspect" genotoxicant.

Key result

Species / strain:

hepatocytes: rat

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the test substance produced a basic pH change at the 166, 500, 1666 and 5000 µg/ml dose levels.

COMPARISON WITH HISTORICAL CONTROL DATA: the negative and positive control values were within the acceptable range of mean historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: all of the concentrations were screened for their toxic effect on the hepatocytes by visual inspection. The dose levels of 1666 and 5000 µg/ml were determined to be cytotoxic and were not evaluated. The highest concentration scored in the assay was 500 ug/ml; the concentration which resulted in detectable abnormal cell morphology. Four other concentrations, 5.0, 16, 50 and 166 µg/ml, were scored as well.

None of the treated cultures produced mean net nuclear grain counts that were substantially greater than the solvent control.

Conclusions:

The test substance was evaluated for genotoxicity in the Rat Hepatocyte Primary Culture/DNA Repair Test. Under the conditions of the assay, the test substance was determined to be negative based upon its inability to produce a mean net nuclear grain count greater than or equal to 5 times the vehicle control at concentrations up to cytotoxic levels (500 µg/ml).

Reference 5

Endpoint:

in vitro transformation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983-10-11 to 1983-11-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well documented non-guideline study, performed under GLP without metabolic activation only. Positive and negative controls were included.

Qualifier:

no guideline available

Principles of method if other than guideline:

Baalb/c-3T3 cells were exposed to the test substance. The cells were then evaluated to determined the test substances ability to induce foci of transformed cells, recognized by dense, piled up colonies on a monlayer of normal cells. This phenomenom is highly correlated with malignant transformations.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): sample No. 5601-22-20; Order No. J-199
- Physical state: light yellow-colored liquid

Species / strain / cell type:

mammalian cell line, other: mouse Balb/3T3 (subclone C-14 of clone 1-13)

Details on mammalian cell type (if applicable):

- Type and identity of media: Cultures were grown and passaged in Eagle's Minimum Essential Medium supplemented with fetal bovine serum, L-glutamine, penicillin and streptomycin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

without

Test concentrations with justification for top dose:

20, 80, 120, 150 and 188 nl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Remarks:

see below

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

not specified

Remarks:

no data provided on the solvent used for the positive control

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

not specified

Remarks:

no data provided on the solvent used for the positive control

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine-1.25 ug/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 72 hours
- Expression time (cells in growth medium): ~4 weeks with refeeding twice a week
- Fixation time (start of exposure up to fixation or harvest of cells): The assay was terminated after the ~ 4 week expression time by fixing the cell monolayers with methanol and staining with Giemsa. The stained dishes were examined by eye and by microscope to determine the number of foci of transformed cells.

NUMBER OF REPLICATIONS: at least 20 dishes

DETERMINATION OF CYTOTOXICITY
- Method: concurrent cytoxicity data was not provided with the transformation assay. However, a preliminary toxicity assay was conducted which measured cytotoxictiy as % relative survival.

OTHER: 24 hours prior to treatment, a series of 60 mm dishes were seeded with 1.0 E 4 cells/dish and incubated.

Evaluation criteria:

Historical experiment with the C-14 subclone of 1-13 cells showed that the distribution of transformed foci was usually symmetric for any given treatment condition. However, large numbers of foci were observed to appear at random in one or more dishes in a set resulting in skewing of the mean number of foci in that set. This skewing appeared to be due to a technical rather than a biological cause and this conclusion was supported by the finding that dishes with high number of foci occurred randomly in all experimental data, in treated dishes as well as in negative controls. The appearance of large numbers of foci in a dish, compared to other dishes in a set, was considered to be caused by mechanical disruption during the refeeding process that occurred twice weekly during the 4-week assay period. Recent analyses of the historical negative control results show that when the data was converted to logarithmic form (base 10) a normal distribution was obtained and a few dishes with abnormally high numbers of foci (e.g. > 10) did not disturb this relationship. With the transformation assay data in a normal distribution, a t-test could be applied for determining statistical significance.
In general, a response at only one dose level just attaining the 95 % confidence level was not considered sufficient evidence of activity in the assay. However, responses at one or more treatment levels attaining the 95 % confidence levels and exhibiting evidence of dependency were considered as positive evidence of transforming activity and responses achieving the 99 % confidence level over one or more test substance treatments were similarly interpreted.

Statistics:

Bailey's modification of Student's t-test was used to determine whether the results for each treatment condition was significantly different from the experimental negative control (~p

Key result

Species / strain:

mammalian cell line, other: mouse Balb/3T3 (subclone C-14 of clone 1-13)

Metabolic activation:

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The test substance was soluble in culture medium forming an alkaline solution at the maximum tested concentration of 74.8 ul/ml. This alkaline stock solution was neutralized (~pH 7.0) using 5M HCl prior to its use in the assay.

RANGE-FINDING/SCREENING STUDIES: The test substance was evaluated in a preliminary cytotoxicity test at concentrations ranging from 0.98 nl/ml to 16 ul/ml in two fold dilution steps. Each dose was applied to three culture dishes seeded 24 hours earlier with 200 cells per dish. After an exposure period of 72 hours, the cells were washed and incubated in growth medium for an additional 3-5 day. The surviving colonies were stained and counted and a relative survival for each dose was obtained by comparing the number of colonies surviving treatment to the colony counts in the negative control dishes. Ten treatments resulted in survivals ranging from 35.5 % at 125 nl/ml to approximately 87.5 to 95.2 % over the 62.5 to 0.98 nl/ml range. No survivors were observed for treatments with 250 nl/ml or higher. The transformation assay is normally applied to concentrations that cause survivals in the 10 % to 100 % range and is considered to be most sensitive near 10-20 % survival, since maximum transformation frequencies for a series of model carcinogens were obtained for treatments that resulted in survivals over this range. Therefore, the concentrations of 188 ul/ml to 20 nl/ml, corresponding to relative survivals of approximately 12 % to nearly 100 % were chosen for the assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No concurrent cytotoxicity evaluation was provided in the study report with the transformation assay. However, a preliminary cytotoxicity test showed relative survivals of approximately 12 % to nearly 100 % were achieved with the doses chosen for the transformation assay.

The negative control measured the frequency of the appearance of spontaneously transformed foci in the current assay. Two transformed foci were observed among the 27 flasks for an average of 0.07 focus/flask. While this spontaneous transformation frequency was within the expected range of 0 -0.5 focus/flask, negative control flasks with high number of transformed foci (>/= 10 foci in a single flask) have been observed in other assays forming the historical negative control data base. The appearance of large numbers of foci in a single flask, compared to the other flasks in a set, was considered to be caused by the respreading of one or a small number of foci by mechanical disruption during the refeeding process that occurred twice weekly for the incubation period. The assumption that the appearance of flasks with high numbers of foci resulted from a technical rather than a biological cause was supported by the finding that such flasks appeared to occur randomly in all experimental data, in treated flasks as well as in negative controls. If such data was included in the analysis of experiments, a marked skewing of the average frequency of foci occurs that is inappropriate for application of parameteric tests for statistical significance. Analyses of the distribution patterns of the transformation data showed that the data closely fit a log-normal distribution curve. Conversion of the raw data to the log 10 resulted in a normal distribution, and the presence of a few flasks with high numbers of foci does not disturb this relationship.

Bailey's modifications of Students' t-test can then be used to avoid the assumptions (intrinsic in many tests of statistical significance) of equal variances and equal numbers of entries in the treatment and control data sets.

Ten positive control treatments with 5.0 ug/ml MCA induced a total of 60 foci among the 18 positive control flasks. Log10 analysis of these data showed that the positive control frequency of 0.589 foci/flask was highly significant (p<0.01) compared to the negative control value of 0.02 focus/flask, and therefore the sensitivity of the assay appeared to be normal.

Sixty one foci were observed among the 18 flasks treated with 1.25 ug/ml MNNG. This result was statically significant (p<0.01) from the negative control, but these data were not used in the evaluation of the performance of this assay, they were not discussed in the text of the report.

After log 10 analysis, the average number of foci/dish ranged from zero at 188, 120 and 20nl/ml to 0.027 at 80nl/ml of the test substance. Compared to the negative control value, none of the frequencies of transformed foci observed for the test substance treatments achieved the 95 % confidence level of being significantly altered. In addition, no evidence of a dose-related response was observed; and therefore, concentrations of text substance from 188 to 20 nl/ml were evaluated as being nontransforming to 3T3 cells.

Conclusions:

The test substance did not induce the appearance of a significant number of transformed foci over the concentration range of 20 to 188 nl/ml. Therefore, the test substance was considered to be inactive in the assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genotoxicity properties of the substance were tested by in vivo mouse micronucleus test according to OECD guideline 474 (Jacob, 1992). The test substance did not cause any substantial increase in the incidence of micronucleated polychromatic erythrocytes or any substantial decrease in the p/n ratio, it is concluded that the test substance did not show any evidence of causing chromosome damage or bone marrow call toxicity when administered orally in the in vivo test procedure.

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:

1992-11-03 to 1992-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Experimental study was run under GLP conditions and according to OECD guidelines.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

equivalent or similar to guideline

Guideline:

other: EEC Annex V Commitee (EEC 1984)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test mtarial (as cited in study report): Texacat ZF-10
- Chemical name: 2-[(2-[2-(dimethylamino)ethoxyethyl)methylamino]ethanol
- Physical appearance: clear colorless liquid
- Analytical purity: 98.3%
- Lot/batch No.: 18610791 (U186-1-0791)
- Expiration date of the lot/batch: February 1994
- Storage condition of test material: room temperature in the dark

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River U.K. Limited, Margate, Kent, UK
- Age at study initiation: 35 days
- Weight at study initiation: 22-24g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Diet (e.g. ad libitum): free access to pelleted Biosure LAD 1 rodent diet
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours per day

Route of administration:

oral: gavage

Details on exposure:

Dose : orally by intragastric gavage
Volume : 20ml/kg bw
Fast period : overnight prior to and for 2 hours after oral dosing.

Duration of treatment / exposure:

Single acute dose

Dose / conc.:

1 408 mg/kg bw/day (nominal)

No. of animals per sex per dose:

40 males and 40 females

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

Mitomycin C, batch number 4530740N, obtained from BDH Limited.
Concentration : 0.6 mg/ml

Tissues and cell types examined:

Bone marrow smear from femus.
Examination of erythrocytes

Details of tissue and slide preparation:

Sampling times : 24, 48 and 72 hours after treatment.

Evaluation criteria:

Evaluation of clinical signs and mortalities
Count of micronucleated polychromatic and normochromatic erythrocytes.
Examination of at least 1000 erythrocytes for each animal and assessment of the ratio of polychromatic to normochromatic erythrocytes

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not valid

Additional information on results:

RESULTS OF PRELIMINARY TOXICITY TEST
PHASE I :
- Dose range: 320, 800, 2000, 5000 mg/kg
PHASE II :
- Dose range: 594, 792, 1056, 1408 mg/kg

Following the dosing in Phase II, any mortality or clinical signs of reaction during the experiment were recorded for a period of 72 hours.
The maximum tolerated dose was estimated to be approximately 1408 mg/kg. This dosage was therfore chosen for use in the micronucleus test.

RESULTS OF DEFINITIVE STUDY
Texacat ZF-10 failed to cause any significant decresae in the ratio of polychromatic to normochromatic erythrocytes.

The positive control with Mitomycin C did not reveal any statistically significant decreases in the ratio of polychromatic to normochromatic erythrocytes. It should be noted that even very cytotoxic compounds such as mitomycine C do not always produce a substantial decrease in this ratio as early as the 24 hours sampling time because of the lag caused by erythrocyte maturation.

Conclusions:

Since the test substance did not cause any substantial increase in the incidence of micronucleated polychromatic erythrocytes or any substantial decrease in the p/n ratio, it is concluded that the test substance did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally in the in vivo test procedure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity - in vitro

- Bacterial reverse mutation assay: The test substance was evaluated for mutagenic potential using the Ames/Salmonella Plate Incorporation Assay in Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA1538 in the absence and presence of metabolic activation (Pharmakon Research International, Inc., 1990). The test substance was negative in all tester strain both in the presence and absence of metabolic activation under the conditions of the study up to a concentration of 10000 µg/plate. The results of other Ames Salmonella/Microsome Plate test were found that the test substance failed to induce reverse mutations in any of the tester strains both in the presence and absence of metabolic activation up to concentration levels of 10.000 ug/plate (Barfknecht et al., 1984).

- Chromosome aberration assay: The test substance was evaluated for induction of chromosome aberrations in primary human lymphocytes treated in the presence of metabolic activation (Adams et al.,1992). The test substance showed some evidence of clastogenic activity at the 15 hour harvest. However, since the results generated from the 10 hour harvest in this study, and the 24 hour harvest from a previous study (no data provided) were clearly negative, the overall conclusion was that the test substance provided an equivocal response in the assay.

- Gene mutation in mammalian cells: No in vitro gene mutation in mammalian cells is not available for the test substance. Data generated with a structurally analogue substance is used for endpoint coverage and a justification for this approach is included in section 13. The substance did not produce any statistically significant increases in the frequency of mutations of CHO cells at concentrations between 0.08 to 0.25% (by volume) in tests without an s9 metabolic activation system (BRRC, 1983). With S9 activation, two tests, with either a similar or narrower range of concentrations as tested without S9, produced no indication of a dose-related, statistically significant effect of the treatments. The test substance was not active in producing gene mutations in CHO cells in these studies. The same is assumed for the test substance.

- In vitro transformation assay: The test substance did not induce the appearance of a significant number of transformed foci over the concentration range of 20 to 188 nl/ml in the mammalian cell transformation assay (Litton Bionetics Inc., 1983). Therefore, the test substance was considered to be inactive in the assay.

- DNA damage and repair : The test substance was evaluated for genotoxicity in the Rat Hepatocyte Primary Culture/DNA Repair Test (Pharmakon Research International Inc., 1985). Under the conditions of the assay, the test substance was determined to be negative based upon its inability to produce a mean net nuclear grain count greater than or equal to 5 times the vehicle control at concentrations up to cytotoxic levels (500 µg/ml).

Genetic toxicity - in vivo

One in vivo mouse micronucleus assay was performed on the substance similar to OECD Guideline 474 and EEC Annex V (Jacob, 1993). Since the test substance did not cause any substantial increase in the incidence of micronucleated polychromatic erythrocytes or any substantial decrease in the p/n ratio, it is concluded that test substance did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally in the in vivo test procedure.

Justification for classification or non-classification

Based on the available data and the criteria laid down in the CLP Regulation (EC)1272/2008, the test substance is considered not to be mutagenic.