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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity:
- in vitro: negative (OECD 471, OECD 476)

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
Study period:
14.10.1980 - 16.10.1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Method: in compliance with Ames B.N.et al., Proc. Nat. Acad. Sci., USA, 70, 2281-2285, (1973) and Mut. Res. 31, 347 -364, (1975)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
4, 20, 100, 500, 2500 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation

Migrated to IUCLID6: 500 µg/plate dissolved in water; for TA100, TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 10µg/plate dissolved in DMSO; for TA100, TA98, TA1538, TA1537, TA1535
Remarks:
with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation

Migrated to IUCLID6: 5 µg/plate dissolved in DMSO; for TA98, TA100, TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylendiamin, 10µg/plate dissolved in DMSO; for TA1538
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-Aminoaeridiniumchloride monohydrate, 100 µg/plate dissolved in DMSO; for TA1537
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h, 37°C

NUMBER OF REPLICATIONS: 4
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium 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 specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results:

Dose µg/plate metabolic activation TA98 TA100 TA1535 TA1537 TA1538
Trial 1 Trial 2 Trial 3 Trial 4 Trial 1 Trial 2 Trial 3 Trial 4 Trial 1 Trial 2 Trial 3 Trial 4 Trial 1 Trial 2 Trial 3 Trial 4 Trial 1 Trial 2 Trial 3 Trial 4
0 + 34 40 31 38 117 112 128 121 10 12 13 12 5 5 6 6 20 18 23 16
4 + 32 34 38 35 115 127 127 113 14 11 13 10 2 5 7 4 24 23 - 21
20 + 38 28 35 32 119 129 114 116 10 15 13 8 3 5 - 4 18 24 17 23
100 + 41 29 43 37 107 125 121 107 13 12 12 13 5 6 6 7 16 11 22 14
500 + 42 33 44 40 111 124 114 117 11 12 13 18 4 4 6 5 20 16 20 19
2500 + 28 30 41 45 143 98 129 113 18 13 7 11 7 4 7 5 18 25 25 24
                       
10 2-Aminoanthracene + 870 910 830 925 1740 1830 1650 1550 225 197 194 203 149 128 140 157 230 269 177 165
500 Cyclophosphamid +         221 219 213 218 132 122 126 106                
                     
0 - 25 24 20 28 122 149 133 114 13 9 13 11 6 7 4 5 12 10 14 15
4 - 23 28 29 24 126 122 138 114 13 8 10 10 6 7 5 4 11 12 11 10
20 - 28 27 24 26 129 115 124 120 8 13 9 10 7 5 8 4 14 12 9 12
100 - 22 33 21 27 123 127 108 141 11 11 14 14 5 5 4 5 13 11 12 17
500 - 22 31 25 25 120 134 103 117 10 16 14 18 7 5 7 4 11 13 11 11
2500 - 25 23 20 23 107 - 138 135 15 14 13 15 4 3 6 3 11 12 12 13
                     
5 MNNG - 1640 1770 1350 1480 2800 3200 2300 2700 2800 2750 2650 2450        
10 4-Nitro-o-phenylendiamin -               263 278 246 250
100 9-Aminoacridiniumchloride monohydrate -                         287 258 263 309        
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT-locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
1st Experiment
without S9 mix (4-hour exposure period)
0; 6.25; 12.5; 25.0; 50.0; 100.0; 150.0; 200.0 µg/mL
with S9 mix (4-hour exposure period)
0; 1.25; 2.5; 5.0; 10.0; 15.0; 20.0; 30.0 µg/mL
2nd Experiment (requirements of the OECD Guideline 476 were failed)
without S9 mix (24-hour exposure period)
0; 6.25; 12.5; 25.0; 50.0; 100.0; 150.0; 200.0 µg/mL
with S9 mix (4-hour exposure period)
0; 1.25; 2.5; 5.0; 10.0; 15.0; 17.5; 25.0 µg/mL
3rd Experiment
without S9 mix (24-hour exposure period)
0; 12.5; 25.0; 50.0; 100.0; 200.0; 300.0; 400.0 µg/mL
with S9 mix (4-hour exposure period)
0; 0.63; 1.25; 2.5; 5.0; 10.0; 15.0; 17.5; 20.0 µg/mL
Vehicle / solvent:
culture medium (Ham's F12)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Details on test system and experimental conditions:
After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.
Statistics:
Due to the clearly negative findings, a statistical evaluation was not carried out.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions chosen, the conclusion is drawn that Propargyl alcohol highly conc. is not a mutagenic substance in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.
Executive summary:

The substance Propargyl alcohol highly conc. was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations.

In this study, in the 1st and 3rd Experiment in the absence and presence of metabolic activation the highest concentrations tested for gene mutations were clearly cytotoxic.

On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies both either without S9 mix or after adding a metabolizing system in two valid experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance Propargyl alcohol highly conc. is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity:
- in vivo: negative (OECD 474)

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
Study period:
1995-10-04 - 1995-12-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD)
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Remarks:
Dept. of Toxicology, BASF AG
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, WIGA, D-W8741 Sulzfeld, Germany
- Weight at study initiation: about 28 g
- Assigned to test groups randomly: yes, acc. randomization plan prepared with an appropriate computer program
- Housing:individually in Makrolon cages, type M I
- Diet (e.g. ad libitum): ad libitum, standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum, drinking water
- Acclimation period: about one week in Makrolon cages, type M III, in groups of 5 separately according to sex


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

ANALYSIS
- The feed used in the study was assayed for chemical and microbiological contaminants.
- The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Technical Services of BASF Aktiengesellschaft as well as for the presence of germs by a contract laboratory.
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Amount of vehicle (if gavage or dermal): 10 ml
- Concentration of test material in vehicle: 30, 60, 120 mg/ml
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- substance formulations were prepared immediately before administration
- The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment
Duration of treatment / exposure:
single intraperitoneal application
Frequency of treatment:
once
Post exposure period:
24 - 48 h
Remarks:
Doses / Concentrations:
300, 600 1200 mg/kg bw
Basis:
analytical conc.
No. of animals per sex per dose:
5 for all test groups and negative controls;
2 male and 3 female for cyclophosphamide;
3 male and 2 female for vincristine
Control animals:
yes
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneally
- Doses / concentrations: 20 mg/kg bw

vincristine
- Route of administration: intraperitoneally
- Doses / concentrations: 0.15 mg/kg bw
Tissues and cell types examined:
polychromatic and normochromatic erythrocytes of bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity deaths were observed down to a dose of 1250 mg/kg body weight. Therefore, a dose of 1200 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 600 mg/kg and 300 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

Test Group - Sampling time - Dose
1 - 24 h - vehicle control
2 - 48 h - vehicle control
3 - 24 h - 300 mg/kg bw
4 - 24 h - 600 mg/kg bw
5 - 24 h - 1200 mg/kg bw
6 - 48 h - 1200 mg/kg bw
7 - 24 h - 20 mg/kg bw cyclophosphamide
8 - 24 h - 0.15 mg/kg bw vincristine

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by SCHMID, W . (1976, 1977):
- The two femora were prepared from the animals, and all soft tissues were removed.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended.
- 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette . Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
- The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes . They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occur, are also scored.

Evaluation criteria:
- The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
- The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value.
- A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes indicates an influence of the test substance specifically on the bone marrow .
Statistics:
The number of polychromatic micronucleated erythrocytes after test substance treatment was nearly the range of the actual control value and within the historical values .
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: down to a dose of 1,250 mg/kg bw
- Clinical signs of toxicity in test animals: 1/4 animals died

SUMMARY TABLE (MALES + FEMALES) : Polychromatic and normochromatic erythrocytes

Test group Interval: 24 h Interval: 48 h
Polychromatic erythrocytes Normocytes / total amount polychromatic erythrocytes Cells with micronuclei (%) Polychromatic erythrocytes Normocytes / total amount polychromatic erythrocytes Cells with micronuclei (%)
Dose (mg/kg bw) polychromatic normochromatic polychromatic normochromatic
vehicle control 10000 3833 1.5 0 10000 5140 1.5 0
300 10000 4351 0.9 1.4
600 10000 3731 1.8 1.1
1200 10000 3794 1.6 0.8 10000 4672 1.1 0.6
20, cyclophosphamide  5000 2918 18.2** 1.4
0.15, vincristine 5000 3332 82.8** 3.6

Thus, the test substance 3-Methyl-l-butyn-3-ol did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d <= D/4) did not deviate from the solvent control value at any of the sacrifice intervals. Nor were large micronuclei (d > D/4) observed either in the negative control group or in the three dose groups of 3-Methyl-l-butyn-3-ol. No inhibition of erythropoiesis induced by the treatment of mice with 3-Methyl-l-butyn-3-ol was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

CLINICAL EXAMINATIONS

- The single intraperitoneal administration of the vehicle in a volume of 10 ml/kg body weight was tolerated by all animals without any signs or symptoms.

- Doses of 300 mg/kg or 600 mg/kg body weight led to staggering about 30 minutes - 1 hour after 'test substance administration.

- In the 1,200 mg/kg group abdominal position, shallow respiration and a narcotic-like state were observed after about 30 minutes - 6 hours after treatment of the animals.

- Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg bw nor that of vincristine in a dose of 0 .15 mg/kg body weight caused any evident signs of toxicity .

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions chosen here, the test substance 3-Methyl-1-butyn-3-ol has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro was analyzed in a study performed in large part equivalent to OECD guideline 471 (BASF, 1980). Bacteria S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538 were treated with concentrations of 4, 20, 100, 500, 2500 ug/plate with and without metabolic activation by a rat liver S9 mix. As result, no genetic toxicity was observed.

The same negative result was found in a similar study, where bacteria S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538 were treated with a saturated vapor containing 51721 ppm 2-methylbut-3-yn-2-ol with and without metabolic activation by Aroclor 1254-induced rat liver S9 mix (Dow Chemical, 1980).

The substance Propargyl alcohol highly conc. (read across; CAS 107 -19 -7) was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (BASF SE; 2010). A detailed read across justification is added in chapter 13. Three independent experiments were carried out, with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations.

In this study, in the 1st and 3rd Experiment in the absence and presence of metabolic activation the highest concentrations tested for gene mutations were clearly cytotoxic. The test substance did not cause any relevant increase in the mutant frequencies both either without S9 mix or after adding a metabolizing system in two valid experiments performed independently of each other.

To evaluate the genetic toxicity in vivo, a micronucleus test conducted according OECD guideline 474 was taken into account (BASF, 1995). In this study, NMRI mice received a single intraperitoneal injection of 300, 600 1200 mg/kg bw. After the preparation and scoring of the polychromatic and normochromatic erythrocytes of bone marrow, no clastogenic effect nor any impairment of chromosome distribution in the course of mitosis were found (BASF, 1995).

Justification for classification or non-classification

Classification for genetic toxicity is not warranted according to the criteria of Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.