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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471 with 1,3 -BDDMA, GLP, +/- metabolic activation: negative with all strains tested. Klimisch score: 1 (HMRTF, 2021)

OECD 476 with 1,3 -BDDMA, GLP, +/- metabolic activation: negative; Klimisch score: 1 (HMRTF, 2021)

OECD 487 with 1,3 -BDDMA, GLP, +/- metabolic acitivation: negative; Klimisch score: 1 (HMRTF, 2021)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04/2020-06/2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 471, GLP.
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 440/2008 B13/14, dated 30-05-2008
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ninth addendum of OECD 471, adopted 21-07-1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TEST MATERIAL:
- Name of Test material: 1,3-BDDMA
- physical state: liquid, colorless
Target gene:
his locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ beta-naphthoflavone induced rat liver S9 Mix
Test concentrations with justification for top dose:
In the pre-experiment the concentration rage of the test item was 3-5000 µg/plate. The pre-experiment is reported as experiment I. Since only slight toxic effects were observed 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
Experiment I (plate incorporation test): 3/ 10/ 33/ 100/ 333/ 1000/ 2500/ 5000 µg/plate
Experiment II (pre-incubation test): 33/ 100/ 333/ 1000/ 2500/ 5000 µg/plate
Vehicle / solvent:
On the day of experiment, the test item was dissolved in DMSO (purity >99%). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. All formulations were prepared freshly before treatment and used within two hours of preparation.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene: with metabolic activation: all strains; 4-nitro-o-phenylene-diamine: without metabolic activation: TA 1537 and TA 98
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation) and pre-incubation

EXPERIMENTAL PERFORMANCE:
For each strain and dose level, including the controls, three plates were used.
Experiment I (Plate incorporation): Test solution at each dose level (solvent or reference mutagen solution (positive control)), S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), bacteria suspension and overlay were mixed in a test tube and poured onto the selective agar plates.
Experiment II (Pre-incubation): The test solution at each dose level, the S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation) and the bacteria suspension were mixed in the test tube and incubated at 37 +/- 1.5°C for 60 minutes.
After the pre-incubation, 2.0 ml overlay agar was added to each tube. The mixture was poured on minimal agar plates. After solidification, the plates were incubated upside down for at least 48 hours at 37 +/- 1.5°C in the dark.
In parallel to each test, a sterile control of the test item was performed and documented in the raw data. Therefore, sterile stock solution, S9 mix/ S9 mix substitution buffer were mixed with 2.0 ml overlay agar and poured on minimal agar plates.
The colonies were counted using the validated computer system, which was connected with a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to the precipitation of the test item the colonies were partly counted manually.

DETERMINATION OF CYTOTOXICITY:
 Pre-Experiment for Toxicity: To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I since the acceptance criteria are met.
Evaluation criteria:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

- regular background growth in the negative and solvent control;
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data;
- the positive control substances should produce an increase above the threshold of twofold (strains TA 98, TA 100, and WP2 uvrA) or threefold (strains TA 1535 and TA 1357) the colony count of the corrresponding solvent control;
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
Statistics:
no appropriate statistical method available
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity observed in Experiment II without metabolic activation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity observed in Experiment I with metabolic activation at 5000 µg/plate and in Experiment II without metabolic activation above 2500 µg/plate and with metabolic activation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
1,3-BDDMA is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of Visiomer (R) 1,3-BDDMA to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.


The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:


Pre-Experiment/ Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate


Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate


The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate in experiment I and at 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate. The undissolved particles had no influence on the data recording.


The plates incubated with the test item showed reduced background growth in experiment I in strain TA 1537 and TA 100 and in experiment II in strain TA 1537, TA 98 and TA 100. 


Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occured in strain TA 100 with S9 mix in experiment I and strain TA 98 and TA 100 in experiment II.  


No substantial increase in revertant colony number of any of the five tested strains was observed following treatment with Visiomer(R) 1,3-BDDMA at any dose, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below generally acknowledged border of biological relevance. 


Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. 


In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base par changes or frameshifts in the genome of the strains used.


Therefore, Visiomer(R) 1,3-BDDMA is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.


 


NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 03 March 2021 - Experimental completion date/ Evaluation completion date: 21 June 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 487, GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 29 July 2016
Deviations:
yes
Remarks:
The test item was handled without light protection (approx.10 min) during weighing. The sponsor confirmed no effect on the stability of the subst. (no exposure to direct sunlight, quite short time of handling). Therefor no affect to validity of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
TEST MATERIAL:
- Name of test material: Visiomer® 1,3-BDDMA
- CAS: 1189-08-8
- EC: 214-711-0
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Blood samples were drawn from healthy non-smoking donors with no known illness or recent exposures to genotoxic agents (e.g.chemicals, ionising radiation) at levels that would increase the background incidence of micronucleate cells. For this study, blood was collected from a male donor (25 years old) for Experiment I, from a male donor (22 years old) for Experiment II and from a female donor (19 years old) for Experiment III.
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9 was used as the metabolic activation system. An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
The protein concentration of the S9 preparation used for this study was 31.0 mg/mL (Lot no. 100920).
Test concentrations with justification for top dose:

PS-Phase separation
With regard to the molecular weight and the purity (90.83%) of the test item, 2202 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 14.3 to 2202 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 76.7 μg/mL and above in the absence of S9 mix and at 134 μg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

Considering the phase separation data of Experiment I, 1500 μg/mL (without S9 mix) were chosen as top concentration in Experiment II.

Due to the steep increase in cytotoxicity, no evaluable concentrations in a cytotoxic range were available. Therefore, the experiment was chosen to be repeated with a modified concentration range with a top concentration of 600 μg/mL (Exp. III).
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine
Details on test system and experimental conditions:
1) Culture conditions
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA 1.5% (v/v) as extract, 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

2) Pre-experiment
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the CBPI in comparison to the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.
This preliminary test was designated Experiment I since the cultures fulfilled the acceptability criteria and appropriate concentrations could be selected for cytogenetic evaluation.

3) Cytogenetic Experiment
a) Pulse exposure
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL culture medium was added. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period. After this period, Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation (Clare et al, 2006, Lorge et al, 2006).

b) Continuous exposure (without S9 mix)
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in and washed with "saline G". The washing procedure was repeated once as described. After washing, the cells were re-suspended in complete culture medium containing 10 % FBS (v/v). Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation (Whitwell et al, 2019).

4) Preparation of cells
The cultures were harvested by centrifugation 40 hrs after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa, mounted after drying and covered with a coverslip.
Evaluation criteria:
Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
(MONC x 1) + (BINC x 2) + (MUNC x 3)
CBPI = ------------------------------------------------
n
CBPI Cytokinesis-block proliferation index
n Total number of cells
MONC Mononucleate cells
BINC Binucleate cells
MUNC Multinucleate cells

Cytostasis % = 100 – 100 [(CBPIT – 1) / (CBPIC – 1)]
T Test item
C Solvent control

Acceptability Criteria:
- The concurrent solvent control will be within the laboratory historical solvent control data range (95% control limit realized as 95% confidence interval)
− The concurrent positive controls should induce responses that are compatible with the lab. historical positive control and produce a statistically significant increase compared with the concurrent solvent control
− Cell proliferation criteria in the solvent control are considered to be acceptable
− All experimental conditions were tested unless one exposure condition resulted in a clearly positive result
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations
− The criteria for the selection of top concentration are consistent with those described in section ‘Dose Selection’
Statistics:
Statistical significance was confirmed by the Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

A linear regression was performed using a validated test script of “R”, to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Both, biological and statistical significance were considered together.
Key result
Species / strain:
lymphocytes:
Remarks:
human
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Exp.I with and without S9 mix, no cytotoxicity was observed up to the highest concentration, which showed phase separation. In Exp. III without S9 mix after continuous treatment, clear cytotoxicity was observed at the highest evaluated concentration.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Due to the steep increase in cytotoxicity, no evaluable concentrations in a cytotoxic range were available. Therefore, the experiment was chosen to be repeated with a modified concentration range with a top concentration of 600 μg/mL (Exp. III).
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: Substance was dermined to be not mutagenic.
Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, Visiomer® 1,3-BDDMA is considered to be non-mutagenic in this in vitro micronucleus test according to OECD 487, when tested up to cytotoxic or phase separating concentrations.
Executive summary:

The test item Visiomer® 1,3-BDDMA, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. The following study design was performed:

Without S9 mix

With S9 mix

 

Exp. I

Exp. II & III

Exp. I

Stimulation period

  48 hrs

48 hrs

  48 hrs

Exposure period

  4 hrs

20 hrs

  4 hrs

Recovery

16 hrs

-

16 hrs

Cytochalasin B exposure

20 hrs

20 hrs

20 hrs

Total culture period

88 hrs

88 hrs

88 hrs

In each experimental group, two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2202 μg/mL of the test item) was chosen with regard to the molecular weight and the purity (90.83%) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487. The rationale for the dose selection is reported in section 3.5.1. The chosen treatment concentrations are reported in Table 1 and the results are summarised in Table 2.

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment III in the absence of S9 mix after continuous treatment, clear cytotoxicity was observed at the highest evaluated concentration.

In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. The mean percentage of the micronuclei in all treated conditions was within the 95% control limits of the historical control data and none of the values were statistically significantly increased, when compared to the solvent control. There was also no concentration related increase in micronucleated binucleates incidence, as judged by an appropriate trend test. The outcome of the study is clearly negative.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Conclusion

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, Visiomer® 1,3-BDDMA is considered to be non-mutagenic in this in vitro micronucleus test according to OECD 487,

when tested up to cytotoxic or phase separating concentrations.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-21-12 to 2021-06-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Kanpoan No. 287 -- Environmental Protection Agency Eisei No. 127 -- Ministry of Health & Welfare Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
TEST MATERIAL:
- Name of test material: 1,3-BDDMA
- CAS No. 1189-08-8
- EC No. 214-711-0
- Storage conditions: at room temperature
- Physical state: colourless liquid
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
According to the OECD Guideline for Cell Gene Mutation Tests at least 4 analysable concentrations should be used in two parallel cultures.
The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.0.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

Main experiment:
without metabolic activation: 15, 30, 60, 120 and 160 μg/mL (exposure time 4 h)
with metabolic activation: 50, 100, 200, 400 and 800 μg/mL (exposure time 4 h)
Vehicle / solvent:
DMSO (purity >= 99.9%)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.7 to 1.2 x 10^7 cells
- After 24 h, the medium was replaced with serum-free medium containing the test item, either without S9 ix or with 50 µl/ml S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was prelaced with complete medium following two washing steps with PBS. Immediately after the end of treatment, the cells were trypsinised and subcultivated. At least 2.0x10^6 cells per experimental point (concentration series plus controls) were subcultivated in 175 cm² flasks containing 30 ml medium. Three to four days after the first subcultivation, at least 2.0x10^6 cells per experimental point were again, subcultivated in 175 cm² flasks containing 30 ml medium. Following the expression time of approx. 7 days, five 75 cm² cell culture flasks were seeded with about 4-5x10^5 cells each in medium containing 6-TG (11 µg/ml). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 +/- 1.5°C in humidified atmosphere with 1.5 +/- 0.5 CO2.
After approx. 8 days (evaluation for viability) and approx. 9 days +/- 2 days (mutation analysis), the colonies were stained with 10% methylene blue in 0.01% KOH solution. Colonies with more than 50 cells were counted. In doubt, the colony sized was checked with a preparation microscope.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survial (RS) as measure of test item induced cytotoxicity. The cultures were incubated at 37 +/- 1.5°C in a humidified atmosphere with 1.5% +/- 0.5 CO2. The colonies to determine the relative survival (RS) were fixed and stained approx. 8 +/- 2 days after treatment.


Evaluation criteria:
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test conditions exhibits a statistically significant increase compared with the concurrent negative control;
b) the increase is dose-related when evaluated with the appropriate trend test;
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
b) there is no concentration-related increase when evaluated with the appropriate trend test;
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.
Statistics:
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies (mean values) obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

A t-test was not performed since all mean mutant frequencies of the groups treated with the test item were well within the 95% confidence interval of our laboratory's historical negative control data.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
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, Visiomer® 1,3-BDDMA is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of Visiomer® 1,3-BDDMA to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
The treatment period was 4 hours with and without metabolic activation.
The maximum test item concentration of the pre-experiment (2202 μg/mL) was chosen with respect to the OECD guideline 476, regarding the purity of the test item.
The maximum concentration of the main experiment was limited by phase separation and cytotoxicity of the test item (maximum concentration 240 μg/mL without metabolic activation) and 1200 μg/mL with metabolic activation). There was no relevant shift of pH value and osmolarity of the medium even at the maximum concentration of the test item.
No substantial and dose dependent increase of the mutation frequency was observed in the main experiment.
The tested concentrations are described in chapter 3.5.2. The evaluated experimental points and the results are summarised in Table 3.
Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.
Conclusion
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, Visiomer® 1,3-BDDMA is considered to be non-mutagenic in this HPRT assay.

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

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

Additional information

For the assessment of the mutagenic potential of 1,3-BDDMA several studies are available: a reverse gene mutation assays in bacteria, a mammalian cell gene mutation assay (HPRT test), an in vitro mammalian cell micronucleus test and an in vitro mammalian cell gene mutation test using the Thymidine kinase gene. 

 

Bacterial reverse gene mutation assays

In a guideline study according OECD 471, S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvr A were exposed to 1,3-BDDMA in DMSO at concentrations of 3/10/33/100/333/1000/2500/5000 µg/plate in a pre-incubation and at concentrations of 33/100/333/1000/2500/5000 µg/plate in a plate-incorporation assay with and without metabolic activation (S9 mix).

1,3-BDDMA was tested up to the top dose mentioned in the OECD test guideline. There was no evidence of induced mutant colonies over background.

The positive controls induced the appropriate responses in the corresponding strains.

This study is considered reliable without restrictions.

  

In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes

In a mammalian cell gene mutation assay according to OECD guideline 476 V79 cells cultured in vitro were exposed to 1,3-BDDMA, in DMSO at concentrations of 50, 100, 200, 400 and 800 μg/mL in the presence of mammalian metabolic activation (S9 mix)and 15, 30, 60, 120 and 160 μg/mL in the absence of mammalian metabolic activation (S9 mix) in two independent experiments.

In the test without S9 mix, 1,3-BDDMA was tested up to cytotoxic concentrations and was found to be not mutagenic. In the test with S9 mix, no cytotoxicity was observed, and the top dose was chosen based on precipitation of test substance. In this experiment with S9 mix, the substance was again found to be not mutagenic.

Therefore, the test substance is considered as non-mutagenic in this HPRT assay.

This study is considered reliable without restrictions.

 

 

In vitro Mammalian cell micronucleus assay

1,3-BDDMA, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. The following study design was performed:

Without S9 mix

With S9 mix

 

Exp. I

Exp. II & III

Exp. I

Stimulation period

  48 hrs

48 hrs

  48 hrs

Exposure period

  4 hrs

20 hrs

  4 hrs

Recovery

16 hrs

-

16 hrs

Cytochalasin B exposure

20 hrs

20 hrs

20 hrs

Total culture period

88 hrs

88 hrs

88 hrs

In each experimental group, two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2202 μg/mL of the test item) was chosen with regard to the molecular weight and the purity (90.83%) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487. The rationale for the dose selection is reported in section 3.5.1. The chosen treatment concentrations are reported in Table 1 and the results are summarised in Table 2.

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment III in the absence of S9 mix after continuous treatment, clear cytotoxicity was observed at the highest evaluated concentration.

In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. The mean percentage of the micronuclei in all treated conditions was within the 95% control limits of the historical control data and none of the values were statistically significantly increased, when compared to the solvent control. There was also no concentration related increase in micronucleated binucleates incidence, as judged by an appropriate trend test. The outcome of the study is clearly negative.

This study is considered reliable without restrictions.


Justification for selection of genetic toxicity endpoint
No single key study has been selected since genotoxicity has to be tested in three different in vitro assays. The substance was found to be not mutagenic in all three in vitro assays.  

 

Compliance to REACH requirements

The in vitro mutagenity test requirements are covered with a bacterial reverse mutation test (OECD 471), an in vitro mammalian cell gene mutation tests using the Hprt and xprt genes (OECD 476), and an in vitro mammalian cell micronucleus test (OECD 487). All mentioned studies are reliable (Reliability 1) and were performed with the substance itself. 

 

 

Justification for classification or non-classification

Based on the available data 1,3-BDDMA is not considered to be mutagenic according to the criteria given in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC. Thus, no labelling is required.