Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames tests on both components AMP and PTSA are negative with and without metabolic activation. AMP is tested negative for mutagenicity in mammalian cells. PTSA did not induce any chromosome aberrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline, GLP study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
Not specified in report
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
uvrA (for WP2 uvrA cultures) and uvrB gene ( for Salmonella cultures), TA-98- his D, TA-100- his G, TA-1535- his G, TA-1537- his C
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli (tester strain WP2uvrA)
Metabolic activation:
with and without
Metabolic activation system:
Acoclor 1254-induced rat liver S9 was used for the metabolic activation system.
Test concentrations with justification for top dose:
100, 333, 1000, 3333, 5000 ug per plate
Vehicle / solvent:
Water was selected as the solvent of choice. The test material was soluble in water at approximately 100ug.mL, the maximum concentration tested.
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: All strains, activated - 2-aminoanthracene ;WP2uvrA, nonactivated - methyl methanesulfonate; TA98, nonactivated - 2-nitrofluorene; TA100, TA1535, nonactivated - sodium azide; TA1537, nonactivated - 9-aminoacridine
Details on test system and experimental conditions:
Salmonella tester strains were recieved directly from Dr. Bruce Ames, University of California, Berkeley, and the E.coli was received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester TA1535 is reverted by mutagens that cause basepair substitutions. Tester TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E.coli is sensitive to basepair substitution mutations, rather than frameshift mutations.To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Each culture wasmonitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 10^9 cells per milliliter.Acoclor 1254-induced rat liver S9 was used for the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single IP injection of Acroclor 1254, 500mg/kg, five days prior to sacrifice. Each batch was assayed for its ability to metabolize.Positive controls, appropriate to the given tester strains, were used as follows:All strains, activated - 2-aminoanthraceneWP2uvrA, nonactivated - methyl methanesulfonateTA98, nonactivated - 2-nitrofluoreneTA100, TA1535, nonactivated - sodium azideTA1537, nonactivated - 9-aminoacridineThe concentration varied according to positive control compound. The sterility of the test article was verified.Preliminary Toxicity AssayTen dose levels of the test article were plated, one plate per dose, with overnight cultures of TA100 and WP2 uvrA on selective minimal agar in the presence and absence of rat liver S9 activation.Mutagenicity AssayAn initial and confirmatory assay was used to evaluate the mutagenic potential of the test material. A minimum of 5 dose levels of the test article, with vehicle and positive controls, were plated with tester strains in the presence and absence of rat liver S9 activation. All dose levels of test article, vehicle controls, and positive controls were plated in triplicate. The test systems were exposed to the test article via the plate incorporation methodology described by Ames, et.al.( (1975) and updated by Maron and Ames (1983). Plates were coded according to the testing laboratory's standard procedures. Plates were incubated for 48-72 hours at 35-39C. Plates not counted immediately following the incubation period were stored at 2-6C until the colonies could be counted.Toxicity and degree of precipation were scored relative to the vehicle control plate using a well-defined 9-point scale. Colonies were counted either by an automated colony counter or by hand unless the assay was preliminary or the plate exhibited toxicity. Plates exhibiting test article precipitate that interferes with an automated colony counter were counted by hand.
Evaluation criteria:
For a positive finding, the test material must cause a dose-related increase in mean revertants per plate of at least one tester strain with a minimum of 2 increasing concentrations of test article. Positive finding for TA1535 and TA1537 is an increase in mean revertants at the peak of dose-response greater than, or equal to, 3x mean control value. Positive finding for TA98, TA100, WP2 uvrA is an increase in mean revertants at the peak of dose-response greater than, or equal to, 2x mean control value.
Statistics:
Mean and standard deviation of the number of revertants per plate were calculated and reported.
Species / strain:
other: S. typhimurium (tester strains TA98, TA100, TA1535, TA1537) and E.coli (tester strain WP2 uvrA)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity AssayThe maximum dose tested was 5000ug/plate, achieved by using a concentration of 100mg/mL and a 50uL plating aliquot. Based on the findings, the maximum dose plated in the mutagenicity assay was 5000ug/plate. Neither precipitate nor appreciable toxicity was observed.Mutagenicity AssayNeither precipitate nor appreciable toxicity was observed in the initial or confirmatory assays. In neither the initial nor confirmatory assay was there a positive response with any of the tester strains at any dose level, in the presence or absence of S9 activation. The mean of each positive control exhibited at least a 3-fold increase in the number of revertants over the mean value of the respective vehicle control, indicating a valid test.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):negativeThe results of this study indicate that, under the conditions of this study, the test material did not cause a positive response with any of the tester strains in the presence and absence of Acrclor-induced rat liver S9.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with full documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no positive controls were used for TA100, TA1535 and TA1537 with metabolic activation. However the number of revertants is very low. Also the number of cells per culture was not specified.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine independent mutant colonies of Salmonella typhimurium
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine deficient
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 rat liver S9-mix
Test concentrations with justification for top dose:
10, 100, 500, 1000 and 5000 micrograms/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: sodium azide (without S9 for TA1535 and TA100; 9-aminoacridine without S9 for TA1537; 2-nitrofluorene without S9 for TA1538 and TA98; and 2-aminofluorene with S9 for TA1538 and TA98.
Remarks:
no positive controls were used used for TA100, TA1535 and TA1537 with metabolic activatiion; however the number of revertants is very low
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Precipitation concentration was >5000 micrograms per plate

Conclusions:
Interpretation of results (migrated information):
negative

The test substance is not mutagenic in the AMES Test
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline, GLP study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Remarks:
Not specified in report
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
0.5, 1.5, 5.0, 15, 50, 150, 500, 1500, 5000 ug/mL
Vehicle / solvent:
sterile distilled water
Untreated negative controls:
yes
Remarks:
sterile distilled water
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
Positive controls:
yes
Positive control substance:
other: Methyl methanesulfonate and 7,12 Dimethyl benz(a) anthracene
Details on test system and experimental conditions:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. A preliminary toxicity assay was used to establish the optimal dose levels for the mutagenesis assay. L5178Y cells were exposed to the vehicle alone and to concentrations of the test article ranging from 0.5-5000 uL in both the absence and presence of S9-activation. Cell population density was determined at 24 and 48 hours after exposure to the test article. Toxicity was measured as suspension of growth relative to the growth of the solvent controls. The mutagenesis assay, initial and independent repeat, was used to evaluate the mutagenic potential of the test article. L5178Y mouse lymphoma cells were exposed to the vehicle alone and 10 concentrations of the test article in both the absence and presence of S9. Positive controls, with and without S9, were tested concurrently. Methyl methanesulfonate was used as the positive control for the non-activated system and 7,12- Dimethyl-benz(a)anthracene was used as the positive control for the S-9 activated system.Tubes containing L5178Y/TK+- cells with F0P medium or S9 activation mixture, and 100uL of test solution at concentrations of 0.5, 1.5, 5.0, 15, 50, 150, 500, 1500, 5000 ug/mL were prepared. Positive controls treated with MMS and 7,12-DMBA were also prepared. Treatment tubes were gassed with CO2 and incubated in the dark for 4 hours. Cells were washed twice, resuspended, gassed with CO2 in air again, and placed on a roller. Cells were counted, and numbers were adjusted if necessary. For expression of TK+- cells, cells were placed in a cloning meduim of granulated agar, mixed with Trifluorothymidine (TFT) or viable count, prewarmed and filled with cloning media. The tubes were centrifuged, supernatant decanted, and cells resuspended in cloning media. Tests were conducted in duplicate. TFT or Viable Count were added to the flask and placed on a shaker. The suspension was placed in petri dishes, placed in cold storage for 30 minutes, then incubated for 10-14 days.Diameters and numbers of colonies were recorded, and analyzed relative to positive and solvent controls. Previous published data indicates that the large colony mutants received localized damage, likely in the form of a point mutation or small deletion within the TK locus, and small colony mutants received damage to collateral loci concordant with the loss of TK activity.
Evaluation criteria:
The result was considered to be a positive repsonse if a concentration-related increase in mutant frequency was observed and one or more dose levels with 10% or greater total growth exhibit mutant frequencies of > or= 100 mutants per 10exp6 clonable cells over background level. A result was considered equivocal if the mutant frequency in treated cultures was between 55 and 99 mutants per 10exp6 clonable cells over background level. Test articles producing fewer than 55 mutants per 10exp6 clonable cells over the background level were concluded to be negative.
Statistics:
Means and standard deviations
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Sterile distilled water was the solvent of choice. The test material was miscible in water at 500 mg/mL, the maximum tested concentration.Preliminary Toxicity AssayThe high dose tested was 5000ug/mL. Osmolality of the solvent control was 300mmol/kg and of the highest dose was 396mmol/kg. Suspension growth relative to the solvent control was 13% at 5000ug/mL without S9 activation, and 31% with S9 activation. Based on these results, the dose levels chosen for the mutagenesis assay ranged from 500-5000ug/mL for both the non-activated and S9-activated cultures.Mutagenesis AssayIn the non-activated system, cultures treated with 1000-500ug/mL were cloned and produced a range in suspension growth of 11-55%. In the S9-activated system, cultures treated with 500-5000ug/mL were cloned and produced a range in suspension growth of 9-98%.No treated cultures exhibited mutant frequencies more than 55 mutants per 10^6 clonable cells over the solvent control. A dose-response trend was not observed in either the activated or non-activated systems. Over the concentration ranges, total growth for the non-activated cultures ranged from 12-60%, and 10-137% for the S9-activated cultures. Results of the independent repeat assay are similar (12-62% and 32-124% for non-activated and S9-activated system suspension growth, respectively; 14-67% and 29-191% for non-activated and S9-activated system total growths, respectively). There was likewise no increase in mutant frequencies over the solvent control. Colony sizing for the positive control yielded the expected increase in small colonies, thereby verifying the methods used to detect small colony mutants.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):negativeThe results of the L5178Y/TK+- Mouse Lymphoma Mutagenesis Assay indicate that, under the conditions of this study, 2-amino-2-methyl-1-propanol did not cause a positive response in the non-activated and S9-activated systems, and was concluded to be negative.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 10-12, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with full documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 fraction following Aroclor 1254 exposure
Test concentrations with justification for top dose:
200, 600, 1902 micrograms per milliliter
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate without S9 and cyclophosphamide with S9
Remarks:
solvent was xylene
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mitotic Index: Concentration related plating efficiency was established in 1000 cells from each of two slides per test group. No influence on mitotic index was observed.

The precipitation concentration was > 1902 ug/mL

Conclusions:
Interpretation of results (migrated information):
negative

The test substance does not induce chromosome mutations (aberrations) in V79 Chinese Hamster cells. The substance is neither mutagenic nor cytotoxic under the conditions of the study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test article-treated groups was not statistically increased relative to their respective vehicle control in either male of female mice, regardless of dose level (maximum dose 60 mg/kg bw AMP) or bone marrow collection time.

No in vivo data are available for PTSA.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline, GLP study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
Not specified in report
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:
ICR mice from Harlan Sprague-Dawley, INC, were 6-8 weeks old at study initiation. For the pilot, toxicity, and MNT assay, males ranged in weight from 27.4g to 31.9g, and females ranged from 23.0g to 28.0g. After arrival to the lab, the animals were monitored for parasites and infections, and were quarantined a minimum of 5 days. They were observed daily for general health, food and water consumption patterns, and other conditions. The animals were deemed healthy prior to placement on study.Mice were housed in an AALAC-accredited facility with appropriate temperature, humidity, and photocycle for the species. They were housed up to 5 per cage, and given food and water ad libitum. The feed and water contained no contaminants that influenced the study.The mice were randomized and placed into assigned dose groups, and were identified by a uniquely numbered ear tag.
Route of administration:
intraperitoneal
Vehicle:
water
Details on exposure:
In each the Pilot & Toxicity Studies (designed to set appropriate dose levels for the micronucleus assay), and the Micronucleus Assay, mice were weighed immediately prior to dose administration and the dose volume was based on individual body weights. Mice were given a single IP injection of the test article in water, the vehicle alone, or the positive control substance.Pilot Study: 2 male mice were dosed with 1, 10, 100, or 1000 mg test material /kg body weight, and 5 males and 5 females were dosed with 2000 mg/kg. Toxicity Study:5 male and 5 female mice were dosed with 200, 400, 600, 800 mg/kg
Duration of treatment / exposure:
Single IP injection
Frequency of treatment:
Once
Post exposure period:
24 and 48 hours
Remarks:
Doses / Concentrations:200, 400, 600, 800 mg AMP/kgBasis:nominal conc.
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes, cyclophosphamide
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
At the scheduled sacrifice times (24 and 48 hours), up to 5 mice / sex / dose were sacrificed, the femurs exposed, and bone marrow aspirated into a syringe of fetal bovine serum. The bone marrow cells were pelleted by centrifugation and the supernatant drawn off. The cells were resuspended and a small amount of suspension spread onto a glass slide. Two to four slides were prepared per animal. The slides were fixed in methanol, stained, and permanently mounted. Slides were coded and scored blindly. Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei. The number of micronucleated normochromatic 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:
The test article was considered to induce a positive response if a dose-responsive increase in mocronucleated polychromatic erythrocytes was observed and one or more doses were statistically-elevated relative to the vehicle control (p>0.05) at any sampling time.  If a single dose group was significantly elevated without a dose-response trend, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended.  The test article would be considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs noted on the day of dosing included: lethargy in males and females at 60 mg/kg (highest dose).
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Pilot:Mortality occurred within 4 hours following dosing: 5/5 males and 5/5 females at 2000 mg/kg, and 2/2 males at 1000 mg/kg. There were no clinical observations noted.Toxicity:Mortality occurred within 3 days of dosing: 5/5 males and 5/5 females at 400, 600, and 800 mg/kg, and 3/5 males and 5/5 females at 200 mg/kg. Clinical signs included: lethargy in males and females at all dose levels, piloerection in males and females, and crusty eyes in males at 200 mg/kg. The LD50/3 was calculated by probit analysis to be approximately 73.7 mg/kg for male and female mice. Micronucleus Assay:There was no treatment-related mortality at any dose level. Clinical signs noted on the day of dosing included: lethargy in males and females at 60 mg/kg (highest dose). All other mice treated with test material and control articles appeared normal during the study. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test article-treated groups was not statistically increased relative to their respective vehicle control in either male of female mice, regardless of dose level or bone marrow collection time.
Conclusions:
Interpretation of results (migrated information): negativeUnder the conditions of the assay described in this report, 2-amino-2-methyl-1-propanol did not induce a sugnificant 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)

Additional information

Justification for classification or non-classification

Based on the above information on the components, the substance is not to be classified for mutagenicity according to CLP (Regulation EC No 1272/2008).