Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All available in vitro genetic toxicity study results were negative.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Clear colourless liquid, purity 96.5%.
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:
S9-mix (Aroclor-1254 induced rat liver S9-mix)
Test concentrations with justification for top dose:
Dose range finding test conducted at 3, 10, 33, 100, 333, 1000, 3330 and 5000 ug/plate. In the dose range finding study, pentamethyl-trioxepane did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Mutation assay and follow up repeat study was conducted at 100, 333, 1000, 3330 and 5000 ug/plate.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
All incubations were carried out in the dark at 37 +/- 1.5 oC for 48 hours.

In the dose range finding test TA100 adn WP2uvrA were exposed to eight different doses with and without 5% S9 mix in triplicate. In the mutation assay, 5 different doses were tested with and without 5% S9 mix in triplicate in each cell line. Frequency of dosing was once.
Evaluation criteria:
- the negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain
- the positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range for each positive control substance. The mean plate count should be at least three times the concurrent vehicle control group mean.
- The selected dose range should include a clearly toxic conc. or should exhibit limited solubility as demonstrated by the preliminary toxicity range finding test or should extend to 5 mg/plate.
Statistics:
Not applicable: negative result
Key result
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
Key result
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
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
Additional information on results:
All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

Not cytotoxic in the dose range finding test or mutation assay, highest conc. 5 mg/plate

Not genotoxic effects in the dose range finding test or mutation assay, highest conc. 5 mg/plate

Pentamethyl-trioxepane precipitated in the top agar layer at conc of 3330 and 5000 μg/plate. At the start of the incubation period precipitation was seen in the 5000 μg/plate. At the end of the incubation period it was gone.

Additionally, the stability of pentamethyl-trioxepane in dimethyl sulfoxide (DMSO) was determined. Conclusion: a 4.22% solution of pentamethyl-trioxepane in dimethyl sulfoxide (DMSO) is completely stable at room temperature during at least 24 hours.

The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The selected dose range extended to 5mg/plate.

Conclusions:
Pentamethyl-trioxepane is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay
Executive summary:

Pentamethyl-trioxepane is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
There was one deviation from the test protocol. The study integrity was not affected. The S9 mix in the second cytogenic assay was prepared using Phenobarbital and β-naphtoflavone instead of Arochlor 1254.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Clear colourless liquid, purity 96.5%.
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Cultured peripheral human lymphocytes were used

Blood was taken from non-smoking male volunteers in December 2004. Dose range finding study: age 35, Average Generation Time (AGT)=14.5h. First cytogenic assay: age 32, AGT=14.2h. Second cytogenic assay: age 39, AGT 14.4h
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-1254 or phenobarbital and beta-naphthoflavone induced rat liver S9-mix
Test concentrations with justification for top dose:
Dose range finding test: 33, 100, 333, 1000 and 1742 μg pentamethyl-trioxepane/ml culture medium with and without S9 mix. First and second cytogenetic assay: 333, 1000, 1742 μg pentamethyl-trioxepane/ml culture medium with and without S9 mix.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Immediately after blood collection lymphocyte culture was started. All incubations were carried out in a humid atmosphere (80-100%), 5 ± 0.5% CO2 in air in the dark at 37 ± 1 °C.

Dose range finding test: cells were cultured for 48h. They were exposed to pentamethyl trioxepane for 3, 24 or 48 h without S9 mix and 3h with S9 mix. Cells were fixated, slides were prepared and cytotox was determined with mitotic index.
First cytogenetic assay: cells were cultured for 48h. They were exposed to pentamethyl trioxepane for 3h without S9 mix and 3h with S9 mix. Cells were fixated and slides were prepared.

Fixation:
Without metabolic activation:
3 h exposure, 24 h fixation
24 h exposure, 24 h fixation
48 h exposure, 48 h fixation
With metabolic activation:
3 h exposure, 24 h fixation
3 h exposure, 48 h fixation

Frequency of dosing: once

Additionally, the stability of pentamethyl-trioxepane in dimethyl sulfoxide (DMSO) was determined. Conclusion: a 4.22% solution of pentamethyl-trioxepane in dimethyl sulfoxide (DMSO) is completely stable at room temperature during at least 24 hours.
Evaluation criteria:
• The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range: Yes
• The positive control substances should produce a statistically significant increase in the number of cells with chromosome aberrations: Yes
• A homogenous response between the replicate cultures is observed: Yes
• A possible precipitate present on the slide should not interfere with the scoring of the chromosome aberrations: no precipitate

A test substance in considered positive if:
• It induced a dose-related statistically significant increase in the number of cells with chromosome aberrations
• A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose response relationship.

A test substance is considered negative in the test if none of the tested conc. induced a statistically significant increase in the number of cells with chromosome aberrations.
Statistics:
Chi-square Test
Key result
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes were used
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
Additional information on results:
Dose range finding test: Result: 0.01 M highest conc recommended for cytogenetic assay.

Not cytotoxic in the dose range finding test or cytogenetic assay, highest conc. 1742 μg pentamethyl-trioxepane/ml culture medium.

No genotoxic effects in the dose range finding test or cytogenetic assay 1742 μg pentamethyl-trioxepane/ml culture medium

Remarks

 

Frequency of reversions/mutations/aberrations, polyploidy as appropriate:

Both in the presence and absence of S9 mix pentamethyl-trioxepane did not induce statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. Also no effects were seen on the number of polyploidy cells and cells with endoreduplicated chromosomes.  The positive control chemicals both produced statistically significant increases in the frequency of aberrant cells. The number of cells with chromosome aberrations found in the solvent control studies was within the laboratory historical control data range.

Precipitation concentration if applicable:

At the highest concentration no precipitation was shown.

Mitotic index:

Number of metaphase was determined per 1000 cells. The mitotic index of pentamethyl-trioxepane in the dose range finding test, first and second cytogenetic assay did not show an effect on the mitotic processes and cell cycle progression as compared with the negative controls. 

Conclusions:
Pentamethyl-trioxepane is not clastogenic in human lymphocytes under the experimental conditions described in this study.
Executive summary:

Pentamethyl-trioxepane is not clastogenic in human lymphocytes under the experimental conditions described in this study.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 14 March 2018 Experimental Completion Date: 11 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)
Specific details on test material used for the study:
Identification: 3,3,5,7,7-pentamethyl-1,2,4-trioxepane
CAS Number: 215877-64-8
Sponsors Identification: Tx 311
Product name: Trigonox 311
Description: Colorless clear liquid
Purity/concentration: 97.9% 3,3,5,7,7-pentamethyl-1,2,4-trioxepane, no correction for purity was made.
Batch Number: 1711423006
Date of Expiry: 20 December 2019
Storage conditions: Room temperature, in the dark
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cell Line
The L5178Y TK +/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit in Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time. The L5178Y cell line has successfully been used in in vitro experiments for many years. L5178Y cells are characterized by a high proliferation rate (doubling time 10 - 12 h in stock cultures) and cloning efficiency of untreated cells of usually more than 50 %, both characteristics are necessary for the appropriate performance of the study. The stock of cells is stored in liquid nitrogen at approximately -196 °C. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Culture
The culture medium used was RPMI 1640 with Glutamax-l and 20mM HEPES buffer supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/mL), Sodium Pyruvate (1mM) and Amphotericin B (2.5 μg/mL) giving R0 media.
For use, a sample of cells were removed from cryogenic storage before the start of the study and grown in R0 media supplemented with 10% horse serum (known as R10 media) at approximately 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and are sub-cultured accordingly. R10 media was used for general maintenance of cells and cell washing at the end of the test item exposure period. R20 media, (R0 with 20% horse serum) was used for the seeding of viability and mutant frequency plates and for maintenance of cultures during the expression period. The serum concentration during the exposure period was 5% for the 4-hour groups and 10% for the 24 hour group.

Cell Cleansing
TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before a stock of cells is frozen these homozygous mutants (TK -/-) must be removed. The cells are cleansed of mutants by culturing in THMG medium for 24 hours. This medium contains Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells are cultured in THG medium (THMG medium without Methotrexate) before being returned to R10 media. The "cleansed" cells are frozen in 1 ml aliquots at between 1 to 3 x 10^6 cells/mL and stored in liquid nitrogen freezers at approximately –196 °C. A fresh ampoule is removed from the frozen stock and cultured to provide adequate numbers of cells for testing.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test
0, 3.40, 6.80, 13.59, 27.19, 54.38, 108.75, 217.5, 435 and 870 µg/mL

Mutagenicity Test
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 2000 μg/mL or 10 mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce approximately 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).

The dose levels plated out for expression of mutant colonies were as follows:
4-hour without S9 - 54.88, 108.75, 217.5, 435, 652.5, 870 μg/mL
4-hour with S9 (2%) - 27.19, 54.88, 108.75, 217.5, 435, 652.5 μg/mL
24-hour without S9 - 27.19, 54.88, 108.75, 217.5, 435, 652.5 μg/mL
Vehicle / solvent:
Following solubility checks performed in-house, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
400 μg/mL and 150 μg/mL for 4-hour and 24-hour exposures respectively
Positive control substance:
ethylmethanesulphonate
Remarks:
Absence of metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
1.5 μg/mL
Positive control substance:
cyclophosphamide
Remarks:
Presence of metabolic activation
Details on test system and experimental conditions:
Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 3.4 to 870 μg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 10^5 cells/mL.
The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post Concentration toxicity, and a comparison of each Concentration SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

Mutagenicity Test
Main Experiment
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. For the 4-hour exposure groups both with and without metabolic activation, the cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. In the 24-hour exposure in absence of metabolic activation the cells were processed to give 0.3 x 10^6 cells/mL in 10 mL cultures established in 25 cm2 tissue culture flasks. The Concentrations were performed in duplicate (A + B), at 8 dose levels of the test item (13.59 to 870 μg/mL in all three exposure groups), vehicle and positive controls. To each universal was added 2 mL of S9-mix (2%) if required, 0.2 mL of the concentration dilutions, (0.15 or 0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL (R10 is used for the 24-hour exposure group). The Concentration vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Measurement of Survival, Viability and Mutant Frequency
At the end of the Concentration period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post Concentration toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate Scoring
Microtiter plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for approximately two to three hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-170 x 10^-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should ideally be within this range. Experiments where the vehicle control values are markedly greater than 200 x 10^-6 mutant frequency per survivor are not normally acceptable and will be repeated.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
Optimum toxicity is approximately 20% survival (80% toxicity), but no less than approximately 10% survival (90% toxicity). RTG values are usually the primary factor to designate the level of toxicity. However, under certain circumstances, %RSG values may also be taken into account. Dose levels that have RTG survival values markedly less than 10% are excluded from any statistical analysis..
For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The Global Evaluation Factor (GEF) value was set at 126 x 10^-6 for the microwell method. Any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Cytotoxicity Test
In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of moderate reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was evidence of marked reductions of %RSG values of cells treated with test item. In all three exposure groups precipitate of the test item was observed at 870 μg/mL. In the subsequent mutagenicity experiments the maximum dose was set at the maximum achievable dose level of 870 μg/mL.

Mutagenicity Test

4-hour exposure in the absence and presence of metabolic activation

There was evidence of slight toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values.
There was no evidence of marked reductions in viability (%V); therefore indicating that residual toxicity had not occurred. Precipitate was observed at 870 μg/mL in the absence of metabolic activation. Precipitate was observed at 652.5 and 870 μg/mL in the presence pf metabolic activation. The excessive precipitate observed at 870 μg/mL in the presence of metabolic activation resulted in this dose level not being plated for viability or 5-TFT resistance.
Neither of the vehicle control mutant frequency values were outside the normal in-house range of 50 to 170 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant increases in the mutant frequency in either of the exposure groups. The GEF value was not exceeded at any dose level.

24-hour exposure in the absence of metabolic activation

There was evidence of marked toxicity following exposure to the test item as indicated by the %RSG and RTG values. There was no evidence of reductions in viability (%V), therefore indicating that residual toxicity had not occurred in this exposure group. Optimum levels of toxicity were achieved at 652.5 μg/mL based on the %RSG value. The excessive toxicity observed at and above 870 μg/mL resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with the positive control substance. Precipitate was not observed in the 24-hour exposure group.
The 24-hour exposure without metabolic activation (S9) Concentration, demonstrated that the extended time point had a marked effect on the toxicity of the test item. The vehicle control mutant frequency was within the normal range of 50 to 170 x 10^-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.
The test item did not induce any toxicologically significant increases in the mutant frequency in the 24-hour exposure in the absence of metabolic activation. The GEF value was not exceeded at any test item dose level, and all of the mutant frequency values were within the acceptable range for a vehicle control.

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 3.4 to 870 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

 

Dose

(mg/mL)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

3.40

85

90

111

6.80

84

81

108

13.59

81

81

114

27.19

83

89

102

54.38

91

97

95

108.75

95

91

98

217.5

75

83

60

435

80

73

27

870

60 p

65 p

2 p

p = precipitate observed at the end of exposure

Summary of Results

 

Main Experiment

Concentration

(µg/mL)

4-Hours-S9

Concentration

(µg/mL)

4-Hours+S9

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

0

 

100

1.00

158.36

 

0

 

100

1.00

116.93

 

13.59

Ø

107

 

 

 

13.59

Ø

99

 

 

 

27.19

Ø

97

 

 

 

27.19

 

90

0.72

116.61

 

54.38

 

93

1.03

143.07

 

54.38

 

90

0.77

134.00

 

108.75

 

99

1.02

140.60

 

108.75

 

97

0.81

139.35

 

217.5

 

85

0.85

166.40

 

217.5

 

85

1.03

113.56

 

435

 

92

0.91

151.55

 

435

 

81

0.73

137.69

 

652.5

 

92

0.84

183.93

 

652.5

 

91

0.77

150.96

 

870

 

73

0.79

146.69

 

870

Ø

77

 

 

 

MF threshold for a positive response = 284.36

MF threshold for a positive response = 242.93

Positive control

 

 

Positive control

 

 

EMS

 

 

 

 

 

CP

 

 

 

 

 

400

 

75

0.60

1133.94

 

1.5

 

77

0.54

731.43

 

 

 

 

 

 

 

 

 

 

 

 

 

               

Concentration

(µg/mL)

24-Hours-S9

 

%RSG

RTG

MF§

0

 

100

1.00

167.39

 

13.59

Ø

103

 

 

 

27.19

 

96

0.95

168.14

 

54.38

 

94

0.93

166.27

 

108.75

 

84

0.93

145.23

 

217.5

 

58

0.79

153.14

 

435

 

27

0.45

155.59

 

652.5

 

10

0.25

145.03

 

870

Ø

3

 

 

 

MF threshold for a positive response = 293.39

Positive control

 

 

EMS

 

 

 

 

 

150

 

52

0.51

1203.85

 


 

Conclusions:
The test item, 3,3,5,7,7-pentamethyl-1,2,4-trioxepane (CAS number 215877-64-8) did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic in this assay.
Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

 

Methods…….

One main experiment was performed. In the main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at up 8 dose levels, in duplicate, together with vehicle (DMSO) and positive controls using 4‑hour exposure groups both in the absence and presence of metabolic activation (2% S9) and a 24-hour exposure in the absence of metabolic activation.

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for expression of mutant colonies were as follows:

 

Main Experiment

Group

Concentration of 3,3,5,7,7-pentamethyl-1,2,4-trioxepane (µg/mL)

 plated for mutant frequency

4-hour without S9

54.88, 108.75, 217.5, 435, 652.5, 870

4-hour with S9 (2%)

27.19, 54.88, 108.75, 217.5, 435, 652.5

24-hour without S9

27.19, 54.88, 108.75, 217.5, 435, 652.5

 

 

Results……..

The maximum dose level analyzed was limited by precipitate of test item in the 4 hour exposure groups. In the 24-hour exposure group the maximum dose level analyzed was limited by test item induced toxicity. At the end of exposure a precipitate of the test item was observed at 870 in the 4-hour –S9 exposure group. A precipitate of the test item was observed at 652.5 and 870 in the 4-hour +S9 exposure group. No precipitate was observed at the end of exposure in the 24-hour exposure group. The vehicle (DMSO) controls had acceptable mutant frequency values that were within the acceptable range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

 

Conclusion

The test item 3,3,5,7,7-pentamethyl-1,2,4-trioxepane (CAS number 215877-64-8), did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in this assay.

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

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus study was conducted and was negative.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Due to cleaning procedures or performance of functional observations in the room, temporary deviations from the level of humidity (with max 20%) occurred. Based on lab historical data these deviations are considered not to affect the study integrity.
GLP compliance:
yes
Type of assay:
other: mammalian germ cell cytogenic assay: micronucleus
Specific details on test material used for the study:
Clear colourless liquid, purity 96.5%.
Species:
mouse
Strain:
NMRI
Remarks:
NMRI BR (SPF)
Details on species / strain selection:
The mice selected (NMRI BR (SPF) are recommended by international guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Females were nulliparous and non-pregnant. The animals were provided by Charles River, Sulzfeld, Germany.

Young adult animals were selected (6-8 weeks old). The body weights of the mice at the start of the treatment were within 20% of the sex mean.

Conditions:
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21 +/- 3 oC and a relative humidity of 30-70%. The room was illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.

Accomodation:
Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing Woody Clean bedding (Woody-Clean type 3/4: Tecnilab-BMIBV, Someren, The Netherlands). Paper bedding was provided as nest material (B.M.I. Helmond, The Netherlands). Acclimatization period was at least 5 days before start of treatment under laboratory conditions.

Diet:
Free access to standard pelletized laboratory animal diet (code VRF 1, Altromin, Lage, Germany).

Water:
Free access to tap water.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The mice received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of Pentamethyl-trioxepane. The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.

Feed was withheld 3-4 h prior to dosing.

The dosing volume was 10 ml/kg body weight. The route and frequency of administration and the volume administered of the negative and positive control was the same as those of the test article.

The systemic toxic signs were recorded at least once a day. The time of death was recorded as precisely as possible. The animals were weighed just prior to dosing.
Duration of treatment / exposure:
single treatment via oral intubation, exposure period/sampling time 24 hrs, with an additional one at 48 hrs for high dose (1000 mg/kg bw male, 750 mg/kg bw female)
Frequency of treatment:
once
Post exposure period:
24hrs and 48 hrs for high dose groups (1000 mg/kg bw male, 750 mg/kg bw female)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
male
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
male
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
male
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
female
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Remarks:
female
Dose / conc.:
190 mg/kg bw/day (actual dose received)
Remarks:
female
No. of animals per sex per dose:
five
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CP; CAS 50-18-0; Endoxan, Asta-Werke, Germany) dissolved in physiological saline (B. Braun, Melsungen, Germany) dosed as a single oral intubation of 50 mg/kg body weight.
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
Bone marrow samples: femurs were removed from the animals and flushed with 2 ml fetal calf serum. The cell suspension is collected and centrifuged at 1000 rpm for 5 min. supernatant was removed with a Pasteur pipette. One drop of serum was left. One drop of cell suspension is placed on a slide. 2 slides were prepared per animal. Slides were stained using the “Wright-stain procedure” in an “Ames” HEMA-tek slide stainer. Slides were scored at 1000x magnification.
Evaluation criteria:
The polychromatic/normochromatic ratio was determined by counting and differentiating the 1st 1000 erythorcytes at the same time. 2000 micronucleated polychromatic erythrocytes were examined.
Statistics:
The polychromatic/normochromatic ratio was determined. Averages and standard deviations were calculated. A wilcoxon rank sum test; two sided test at p < 0.05 was used to test if there was a significant increase in micronucleated polychromatic erythrocytes.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Cyclophosphamide induced a significant increase in the number of micronucleated polychromatic erythrocytes and a decrease in the polychromatic to normochromatic erythrocytes ratio (= toxic effects on erythropoiesis). The animals treated with pentamethyl-trioxepane and the negative control showed no increase in the number of micronucleated polychromatic erythrocytes and no decrease in the polychromatic to normochromatic erythrocytes ratio.

During the first hours the highest dose groups showed ataxia and increased activity. Within 18 hours the animals recovered.
Conclusions:
pentamethyl-trioxepane is not mutagenic in the micronucleus test under the experimental conditions described in this report.
Executive summary:

pentamethyl-trioxepane is not mutagenic in the micronucleus test under the experimental conditions described in this report.

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

Additional information

Justification for classification or non-classification

All available in vitro and in vivo genetic toxicity study results are negative. Data are conclusive, but not sufficient for classification.