Reaction products of 2,2-dimethylpropane-1,3-diol with 1-choloro-2,3-epoxypropane

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 June 2020

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane

EC Number:

701-333-0

Molecular formula:

C11H20O4

IUPAC Name:

Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane

Details on test material:

It is a UVCB substance.

Specific details on test material used for the study:

CAS number: 17557-23-2

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

5.5 Metabolic Activation System
The test bacteria were also exposed to the test item in the presence of an appropriate metabolic
activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

5.5.1 Rat Liver S9 Fraction
The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was
provided by Trinova Biochem GmbH, Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA). Certificate of
Analysis was obtained from the supplier. The copies of the corresponding Quality Control &
Production Certificates of the S9 fractions are collected in the raw data notebook and are
attached as Appendix VII. The originals are stored in the Laboratory of TOXI-COOP ZRT.
The following lots of the S9 were applied:
Lot Number: 4303; Expiry date: August 27, 2022; Protein content: 37.6 mg/mL
(used in the initial mutation test);
Lot Number: 4344; Expiry date: October 15, 2022; Protein content: 33.5 mg/mL
(used in both experimental phases of the study);
Lot Number: 4399; Expiry date: January 27, 2023; Protein content: 37.3 mg/mL
(used in both experimental phases of the study)
5.5.2 The S9 Mix (with Rat Liver S9)
Salt solution for S9 Mix Final concentration in S9 Mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 mL
Sterilized by filtration through a 0.22 µm membrane filter.
The complete S9 Mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix 400 mL
The S9 Mix (containing 10 % S9) was kept in an ice bath before it was added to the culture
medium.
5.5.3 Sodium Phosphate Buffer (0.2 M, pH 7.4)
Solution A:
Na2HPO4 x 12H2O 71.63 g
Ultrapure water ad 1000 mL
Solution B:
NaH2PO4 x H2O 27.6 g
Ultrapure water ad 1000 mL
Solution A 880 mL
Solution B 120 mL*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected. The correction
was performed with admixture of the solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm
membrane filter.

Test concentrations with justification for top dose:

Based on the results of the preliminary tests, the test item solutions were prepared from the
test item with dimethyl sulfoxide (DMSO) to obtain the dosing solutions (see: Table 1).
For the selected concentration range, the absent cytotoxicity and the noticed solubility
properties of the test item were taken into consideration, based on the recommendations in
OECD 471 guideline [6]. The recommended maximum test concentration of 5 mg/plate was
chosen as highest test item concentration for planned experiments.
The Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane concentrations investigated in the initial and confirmatory mutation tests:
±S9: 5000, 1600, 500, 160, 50 and 16 µg/plate.
As indicated in section 6.1.2, at the chosen top concentrations no precipitate was expected
on the minimal glucose agar plates.
The test solutions were freshly prepared at the beginning of the experiments. At the
preparation of the test item solutions any correction (multiplier) factor was not taken into
consideration, the doses were based on the final formulation as is.

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other:

Details on test system and experimental conditions:

Origin of the Bacterial Strains
Tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia
coli WP2 uvrA were obtained from:
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures. The identification codes, arrival
date and expiry dates of the actual applied stock cultures are summarized in Table 3.

Rationale for test conditions:

Based on the results of the preliminary tests, the test item solutions were prepared from the
test item with dimethyl sulfoxide (DMSO) to obtain the dosing solutions (see: Table 1).
For the selected concentration range, the absent cytotoxicity and the noticed solubility
properties of the test item were taken into consideration, based on the recommendations in
OECD 471 guideline [6]. The recommended maximum test concentration of 5 mg/plate was
chosen as highest test item concentration for planned experiments.
The Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane concentrations investigated in the initial and confirmatory mutation tests:
±S9: 5000, 1600, 500, 160, 50 and 16 µg/plate.
As indicated in section 6.1.2, at the chosen top concentrations no precipitate was expected
on the minimal glucose agar plates.
The test solutions were freshly prepared at the beginning of the experiments. At the
preparation of the test item solutions any correction (multiplier) factor was not taken into
consideration, the doses were based on the final formulation as is.

Evaluation criteria:

The colony numbers on the untreated, vehicle control, positive control and the test plates
were determined (counted manually, evaluated by unaided eye), the mean values, standard
deviations and the mutation rates were calculated:
Mutation Rate =
Mean number of revertants at the test item (or control*) treatments
Mean number of revertants of vehicle control
* : untreated, vehicle or positive control
A result is considered positive if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the concentration
groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high
as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2
uvrA the number of reversions is at least three times higher than the reversion rate of the
vehicle control.
According to the guidelines, the biological relevance of the results is the criterion for the
interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a concentration-related
increase in the number of revertants nor a reproducible biologically relevant positive
response at any of the concentration groups, with or without metabolic activation.

Statistics:

The colony numbers on the untreated, vehicle control, positive control and the test plates
were determined (counted manually, evaluated by unaided eye), the mean values, standard
deviations and the mutation rates were calculated:
Mutation Rate =
Mean number of revertants at the test item (or control*) treatments
Mean number of revertants of vehicle control
* : untreated, vehicle or positive control

Results and discussion

Test results

Test results 1

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show (see Appendix I to III) that under the
experimental conditions applied, the test item induced gene mutations by base-pair
substitution in the genome of the strains of Salmonella typhimurium TA100, TA1535
and Escherichia coli WP2 uvrA. While the results of the main experiment confirmed well
the concentration range finding test results in Salmonella typhimurium TA100 strain, the
positive results obtained in Salmonella typhimurium TA1535 and Escherichia coli WP2
uvrA strains were not confirmed with a subsequent experiment.
In conclusion, the test item Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane has unequivocal mutagenic activity on strains
carrying base-pair substitution, in Salmonella typhimurium TA100, TA1535 strains in
the absence and presence, in Escherichia coli WP2 uvrA, in the presence of exogenous
metabolic activation, under the test conditions used in this study.

Executive summary:

Purpose of the study:
The test item Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane was tested with regard to a potential mutagenic activity using the
Bacterial Reverse Mutation Assay.
Bacterial strains, exogenous metabolic activation:
The main experiment was carried out using histidine-requiring auxotroph strains of
Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537),
and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2
uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from
livers of Phenobarbital/β-naphthoflavone-induced rats.
Experimental phases:
The study included a preliminary solubility test, a preliminary concentration range finding
test (informatory toxicity test applying the plate incorporation method), and an initial
mutation test (plate incorporation test). Because of the unequivocal, positive mutagenicity
results of the initial mutation test, further testing for the final conclusion was considered not
necessary.
Vehicle, test item concentrations, rationale for concentration selection:
Based on the results of the solubility test and the concentration range finding test the test
item was dissolved in dimethyl sulfoxide (DMSO) and the following concentrations of the
test item were prepared and investigated in the initial mutation test:
±S9: 5000, 1600, 500, 160, 50 and 16 µg/plate.
For the selected concentration range, the absent cytotoxicity, and the noticed solubility
properties of the test item were taken into consideration, based on the recommendations in
OECD 471 guideline [6].
At the preparation of the test item solutions any correction (multiplier) factor was not taken
into consideration, the concentrations were based on the final formulation as is.
Solubility, precipitation:
No precipitation of the test item was observed on the plates in the examined bacterial strains
at any examined concentration level (±S9) throughout the study.
Cytotoxicity results:
In the initial mutation test, no inhibitory effects of the test item on bacterial growth were
observed in the examined strains. All of the noticed lower revertant colony numbers (when
compared to the revertant colony numbers of the corresponding vehicle control) remained
within the range of the biological variability of the applied test system. Furthermore, the
background lawn development was not affected in any case.
Mutagenicity results:
The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO)) plates with
and without S9 Mix demonstrated the characteristic mean numbers of spontaneous
revertants that were in line with the corresponding historical control data ranges.
Electronic copy 1 of 1
Study Number: 555-471-6266
Bacterial Reverse Mutation Assay with Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane page 10 of 31
The reference mutagen treatments (positive controls) showed the expected, biological
relevant increases (more than 3-fold increase) in induced revertant colonies and the number
of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for
the positive control.
In the performed initial mutation test biologically relevant revertant colony number increases
were observed in revertant colony numbers of in case of Salmonella typhimurium TA100,
TA1535 strains in the absence and presence of exogenous metabolic activation (±S9);
furthermore, in Escherichia coli WP2 uvrA in the presence of metabolic activation (+S9).
In these strains the obtained revertant colony number increases showed clear concentration
dependency.
The results of the main experiment confirmed well the concentration range finding test
results in Salmonella typhimurium TA100 strain.
Conclusion:
The reported data of this mutagenicity assay show (see Appendix I to III) that under the
experimental conditions applied, the test item induced gene mutations by base-pair
substitution in the genome of the strains of Salmonella typhimurium TA100, TA1535
and Escherichia coli WP2 uvrA. While the results of the main experiment confirmed well
the concentration range finding test results in Salmonella typhimurium TA100 strain, the
positive results obtained in Salmonella typhimurium TA1535 and Escherichia coli WP2
uvrA strains were not confirmed with a subsequent experiment.
In conclusion, the test item Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane has unequivocal mutagenic activity on strains
carrying base-pair substitution, in Salmonella typhimurium TA100, TA1535 strains in
the absence and presence, in Escherichia coli WP2 uvrA, in the presence of exogenous
metabolic activation, under the test conditions used in this study.