2-Butyne-1,4-diol, polymer with methyloxirane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Bacterial gene mutation (OECD 471, Ames, GLP) : negativ (BASF, 1995) Bacterial gene mutation (OECD 471, Ames, GLP, RA to CAS 32167-31-0) : negativ (BASF, 2012) Bacterial gene mutation (OECD 471, Ames, GLP, RA to CAS 38172-91-7) : negativ (BASF, 1995) Mammalian gene mutation (OECD 476, HPRT, GLP, RA to CAS 38172-91-7 ): negativ (BASF, 2012) Cytogenicity in vitro (OECD 473, Chromosom abberation, GLP, RA to CAS 38172-91-7 ): positiv (BASF, 2012) Cytogenicity in vivo (OECD 474, MN, GLP, RA to CAS 38172-91-7): negativ (BASF, 2013)

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: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: mean value 36.3 g (SD +-1.7 g) first main-experiment; mean value 34.7 g (SD +- 1.9 g) second main-experiment
- Assigned to test groups randomly: [yes]
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: for a minimum of five days after their arrival

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2 °C
- Humidity (%): 35 - 75 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: [water]
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals.
The administered volume was 10 mL/kg b.w. including test substance.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was dissolved in sterile water. The vehicle was chosen due to its relative non-toxicity for the animals. All animals received a single standard volume orally.

Duration of treatment / exposure:

single oral application

Frequency of treatment:

once

Post exposure period:

24 or 48 hours

Remarks:

Doses / Concentrations:
125, 250 and 500 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

7 males for the test substance, 5 males for controls

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration potent inducer of micronuclei
- Doses / concentrations: 40 mg/kg bw.

Tissues and cell types examined:

Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration.
The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of approximately 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test item, the vehicle, or the positive control substance once orally. Seven males were treated per dose group and sampling time. However, one animal of the highest dose group (48 h sampling time) died 48h after administration. Five males each were treated for each vehicle and the positive control group. The animals of all dose groups, except the positive control were examined for acute clinical signs at intervals of around 1 h, 2 - 4 h, 6 h, 24 h, and/or 48 h after administration of the test item and vehicles.
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.

DETAILS OF SLIDE PREPARATION and METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
All animals per sex and test group were evaluated as described.

Evaluation criteria:

The study was considered valid as the following criteria are met:
- at least 5 animals per test group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the vehicle control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the vehicle control.

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test (8)) are used as an aid in evaluating the results, if necessary. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test are used as an aid in evaluating the results, if necessary.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

mortality in one animal at 500 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Two animals of each sex treated in the pre-experiments received the test item 2-Propyn-1-ol, compd. with methyloxirane dissolved in sterile water once orally. The volume administered was 10 mL/kg b.w.. The following dose levels were tested 500, 1000 and 1500 mg/kg bw.
Clincical signs observed: 500 mg/kg bw: Eyelid closure and Ruffled fur; 1000 mg/kg bw: Reduction of spontaneous activity, Eyelid closure and Ruffled fur; 1500 mg/kg bw: Reduction of spontaneous activity, Abdominal position, Eyelid closure, Apathy, Tumbling, Hunchback, Hypothermia, Ruffled fur and Death.
On the basis of these data 1000 mg/kg b.w. were estimated to be suitable as highest dose in the main experiment (which was later reduced due to overt toxicity and death). No substantial sex specific differences were observed with regard to clinical signs. In accordance with the guidelines the main study was performed using males only.


RESULTS OF DEFINITIVE STUDY
In the first main experiment for each test item dose groups 7 males received once orally administrations of 2-Propyn-1-ol, compd. with methyloxirane dissolved in sterile water. The volume administered was 10 mL/kg b.w.. The symptoms of toxicity observed following treatment were: 1000 mg/kg bw: Reduction of spontaneous activity, Abdominal position, Eyelid closure, Ruffled fur, Hunchback and death of 4/7 animals; 500 mg/kg bw: Reduction of spontaneous activity and Ruffled fur; 250 mg/kg bw: Ruffled fur. The animals treated with the negative control (sterile water) did not express any toxic reaction.
High mortality was observed after treatment with the high dose in the 48 hours group (male no. 37, 39, 40 and 41). The animals were found dead in their cage. Due to this mortality, an additional low dose and a high dose group (48 hours post-treatment) were included in a second experiment in order to fulfil the OECD guideline requirements for a valid study.
In the second main experiment for each test item dose groups 7 males received also once orally administrations of 2-Propyn-1-ol, compd. with methyloxirane dissolved in sterile water. The volume administered was 10 mL/kg b.w.. The symptoms of toxicity observed following treatment were: 500 mg/kg bw: Reduction of spontaneous activity, Ruffled fur and death; no clinical signs were observed at 125 mg/kg bw. The animals treated with the negative control (sterile water) did not express any toxic reaction.

Conclusions:

Interpretation of results (migrated information): negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item 2-Propyn-1-ol, compd. with methyloxirane did notinduce micronuclei.

Executive summary:

The test item 2-Propyn-1-ol, compd. with methyloxirane was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was dissolved in sterile water, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

At least five males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

Based

on results of pre-experiments doses of 250, 500 and 1000 mg/kg b.w. were selected for the main experiment. However, due to high mortality observed at 1000 mg/kg (4 of 7 males of the 48 hours group) this dose was not appropriate for evaluation in accordance with the guidelines (less than 5 animals available for evaluation in the 48 hour group and maximum tolerated dose level clearly exceeded). Additional groups of male mice were treated including vehicle and positive control in a second main experiment in order to fulfil the OECD guideline requirements for a valid study. Finally, following dose levels of the test item were investigated and evaluated :

24 h preparation interval: 125^b), 250^a), and 500^a)mg/kg b.w.
48 h preparation interval: 500^b)mg/kg b.w.

                  ^a)First main experiment^b)Second main experiment

In the second main experiment one of 7 males male died after treatment with the high dose (500 mg/kg b.w., 48 hours post-treatment).

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that 2-Propyn-1-ol, compd. with methyloxirane did not exert a cytotoxic effect in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with 2-Propyn-1-ol, compd. with methyloxirane were below or near to the value of the vehicle control groups. Only the micronucleus frequency found in the high dose group (500 mg/kg, 48 hours treatment) was statistically significantly higher (0.108%) compared to the concurrent vehicle control value (0.030%) which was incidentally quite low. However, the micronucleus frequency in the 500 mg/kg (48h) group as well as in all other test item treated dose groups at any preparation interval were very well within the laboratory’s historical vehicle control data (0.010-0.250%, mean 0.108%). Thus, the observed statistical significance at 500 mg/kg (48h) was not considered to have any biological relevance. 

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item 2-Propyn-1-ol, compd. with methyloxirane did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

 Justification for grouping of substances and read-across

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

 

Since only an in vitro gene mutation study in bacteria without E. coli is available for2-Butyne-1,4-diol, polymer with methyloxirane (CAS 61596-96-1),a read-across for in vitro cytogenicity and gene mutation in vitro as in mammalian cells was performed from the structurally related analogue substances 2-Butyne-1,4-diol, comp. with oxirane (CAS 32167-31-0) and 2-Propyn-1-ol, compd. with methyloxirane (CAS 38172-91-7). A summary of the available data on genetic toxicity is given below.

	Target Substance	Source Substance No 1	Source Substance No 2
Name	2-Butyne-1,4-diol, comp. with methyloxirane	2-Butyne-1,4-diol, comp. with oxirane (Polyethylene glycol ether with 2-butyne-1,4-diol)	2-Propyn-1-ol, compd. with methyloxirane
CAS	61596-96-1	32167-31-0	38172-91-7
In vitro gene mutation in bacteria	OECD 471 without E.coli, negative (BASF, 1995) (RL1)	OECD 471, negative (BASF, 2012) (RL1)	OECD 471, negative (BASF, 1995) (RL1)
In vitro gene cytogenicity in mammalian cells	--	--	OECD 473, Positive (BASF, 2012) (RL1)
Mammalian gene mutation	--	--	OECD 476, negative (BASF, 2012) (RL1)
In vivo micro nucleus test	--	--	OECD 474, negative (BASF, 2013) (RL1)

 

The above mentioned substances are considered to be similar on the basis of structural similarity resulting in similar properties and/or activities. The available endpoint information is used to predict the same endpoints for2-Butyne-1,4-diol, polymer with methyloxirane (CAS 61596-96-1).A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

2.    Results

a)    Target substance (CAS 61596-96-1)

In vitro gene mutation in bacteria

The test substance was tested for its mutagenic potential based on the ability to induce back mutations in selected loci in several strains of Salmonella typhimurium in the Ames test according to OECD 471 with and without S9 -mix and under GLP conditions. The used strains were TA 1535, TA 100, TA 1537, TA 98 with dose ranges from 20 µg - 5000 µg/plate with the following results: An increase in the number of his• revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance “confidential TI name” is not mutagenic in the Ames test under the experimental conditions chosen here. (BASF 1995)

 

b)   Source Substance No 1(CAS 32167-31-0)

 

In vitro gene mutation in bacteria

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA were dosed a dose range from 33μg - 5000μg/plate (SPT) and 33μg - 5 000μg/plate (PIT) with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix and a weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2500μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation. (BASF, 2011)

c)    Source Substance No 2(CAS 38172-91-7)

 

In vitro gene mutation in bacteria

The substance Propyn-1-ol, compd. with methyloxirane (a.i. 53.4%) was tested for its mutagenic potential based on the ability to induce back mutations in selected loci in several strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537, TA 98) in the Ames test with a concentration range of 20 µg - 5000 µg/plate with and without metabolic activation according to OECD 471 under GLP conditions with the following results: Toxicity: No bacteriotoxic effect was observed. Mutagenicity: An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substancePropyn-1-ol, compd. with methyloxiraneis notmutagenic in the Ames test under the experimental conditions chosen here (BASF, 1995).

 

In vitro cytogenicity in mammalian cells (CA)

 

The test item 2-Propyn-1-ol, compd. with methyloxirane, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The highest applied concentration (7690μg/ml) was chosen with regard to the composition of the test item and with respect to the current OECD Guideline 473. Duplicates with at least 100 metaphases per culture were evaluated. ln Experiment lA in the absence and presence of S9 mix clear cytotoxicity was observed at the highest evaluated concentration. ln Experiment IB only moderate cytotoxicity was observed in the absence and presence of S9 mix. Statistically significant increases of aberrant cells were observed in Experiment lA in the absence of S9 mix after treatment with 3845.0μg/mL (4.8 % aberrant cells, excluding gaps) and in the presence of S9 mix after treatment with 961.3μg/mL (5.3 % aberrant cells, excluding gaps). ln Experiment IB clastogenicity clearly exceeding the historical solvent control data (0.0- 4.0 % aberrant cells, excluding gaps) was observed at nearly all evaluated concentrations with (8.5 - 14.5 % aberrant cells, excluding gaps) and without (7.0 - 16.0 % aberrant cells, excluding gaps) metabolic activation. No relevant increase in polyploid and endomitotic metaphases was found after treatment with the test item as compared to the frequencies of the control cultures. Appropriate mutagens (EMS and CPA) were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations. ln conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro.

Therefore, 2-Propyn-1-ol, compd. with methyloxirane is considered to be clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to cytotoxic or the highest required concentrations. (BASF, 2012)

In vitro gene mutation in mammalian cells (HPRT)

The test item 2-Propyn-1-ol, compd. with methyloxirane was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD 476.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. In experiment I and II mutant colonies exceeded the range of the historical control data at 7690μg/mL and 961.3 µg/mL. However, the threshold of three times the mutation frequency of the corresponding solvent control was not reached or exceeded. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.8 up to 36.5 mutants per 106 cells; the range of the groups treated with the test item was from 6.5 up to 50.9 mutants per 10^6 cells. EMS (150μg/mL) and DMBA (1.1μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, 2-Propyn-1-ol, compd. with methyloxirane is considered to be non-mutagenic in this HPRT assay. (BASF, 2012)

 

In vivo micro-nucleus test

 

The test item 2-Propyn-1-ol, compd. with methyloxirane was assessed in the micronucleus assay according to OECD 474 for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was dissolved in sterile water, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. At least five males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

Due to a not appropriate dose a second experiment was conducted to fulfill the requirements of the OECD guideline. Finally, following dose levels of the test item were investigated and evaluated: 24 h preparation interval: 125, 250, and 500mg/kg bw. and 48 h preparation interval: 500mg/kg bw.

 

In the second main experiment one of 7 males male died after treatment with the high dose.

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that 2-Propyn-1-ol, compd. with methyloxirane did not exert a cytotoxic effect in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. During the study described and under the experimental conditions reported, the test item2-Propyn-1-ol, compd. with methyloxirane did notinduce micronuclei (BASF, 2013).

 

Key study assignment

 

For genetic mutation in bacteria three studies are available, the most appropriate study is conducted with the target substance and therefore used as key study. For the endpoints mammalian gene mutation and cytogenicity only one study for the source substance 2 are available, hence all these studies are used as key studies. 

Conclusions

Six studies investigating respectively the genetic mutation in bacteria in-vitro, the in-vitro gene mutation in mammalian cells, the in vitro cytogenicity in mammalian cells and the in vivo micro nucleus test are available with the target substance (CAS 61596-96-1) or a read across substance(CAS 32167-31-0 and CAS 38172-91-7).In vitro gene mutation in bacteriaand in vitro gene mutation in mammalian cellswere negative, whilein vitro cytogenicity in mammalian cells was positive. According to ITS an in vivo micro-nucleus test was performed providing negative results.

 

In summary, it could be concluded, that2-Butyne-1,4-diol, polymer with methyloxirane (CAS 61596-96-1) is not mutagenic.

Justification for selection of genetic toxicity endpoint
In vivo study.

Justification for classification or non-classification

Based on the information received for the target substance and structurally related source substances, no classification for the target substance 2-Butyne-1,4-diol, polymer with methyloxirane (CAS 61596-96-1)is derived according to Regulation (EC) 1272/2008 or Directive 67/548/EEC,

GHS: no classification

DSD: no classification