Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria (OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A pKM 101.

In vitro cytogenicity / micronucleus study: Pursuant to ECHA Decision CCH-D-2114465595-37-01/F (issued 4 July 2019) an in vitro micronucleus study according to OECD guideline 487 is currently ongoing. Preliminary results are available, which are negative with and without metabolic activation in human lymphocytes. Nevertheless, based on the final results, the hazard assessment with respect to cytogenicity/ micronuclei formation will be updated accordingly.

In vitro gene mutation in mammalian cells (OECD 476): negative with and without metabolic activation in Chinese hamster ovary (CHO) cells.

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine-dependent auxotrophic mutants
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
other: tryptophan-dependent mutant
Metabolic activation:
with and without
Metabolic activation system:
liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
5, 15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
Polyol R3530 was partly miscible with water. It was found to be miscible at 50 mg/mL with water. Water (purified in-house by reverse osmosis) was,
therefore, used as the vehicle for this study.
The highest concentration of Polyol R3530 tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration as recommended in the regulatory testing guidelines. The highest concentration in each test was diluted with water to produce a series of lower concentrations, separated by approximately half-log10 intervals.
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Sodium azide, 9-Aminoacridine, 2-Nitrofluorene, 4-Nitroquinoline-1-oxide, 2-Aminoanthracene, Benzo[a]pyrene
Details on test system and experimental conditions:
Preparation of S9 fraction
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at approximately -80°C.

Preparation of S9 mix
The S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water. All the cofactors were filter-sterilised before use.

Mutation test procedure: first test (plate incorporation test)
Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 μg/plate), positive control or negative control were placed in glass vessels. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer). Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration selected for use in the second test would be the same as that used in the first. If toxic effects were observed at more than one concentration, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be obtained.

Mutation test procedure: second test (pre-incubation test)
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.

Evaluation criteria:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10E9/mL.
Statistics:
The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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:
Results
The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation.
The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms.
The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory.
Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

First test (plate incorporation test)
No evidence of toxicity was obtained following exposure to Polyol R3530. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Polyol R3530 at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

Second test (pre-incubation test)
Based on the results of the first assay, the highest test concentration was maintained at 5000 µg/plate also for the present assay. No evidence of toxicity was obtained following exposure to Polyol R3530. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Polyol R3530 at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.
Conclusions:
It is concluded that Polyol R3530 showed no evidence of mutagenic activity in bacteria under the test conditions used.
Endpoint:
in vitro cytogenicity / micronucleus study
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:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted in 2016
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Batch No.: P2D4000629
Expiry date: 2020-07-04
Appearance: Yellowish Liquid
Storage conditions:Ambient (15 – 25 °C)
Purity: 100% (UVCB as produced)
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
For lymphocytes:
- Sex, age and number of blood donors: Healthy non-smoking donors, approximately 18 – 35 years of age, not receiving medication. Blood was collected from single donors for each experiment.
- Whether whole blood or separated lymphocytes were used: Whole blood (5 – 12% mixture of whole blood in medium within 30 h after blood collection)
- Whether blood from different donors were pooled or not: Will be specified in the report.
- Mitogen used for lymphocytes: Phytohaemagglutinin (PHA), 48 h exposure

MEDIA USED
- Type and composition of media:
Culture medium: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1), supplemented with 200 mM GlutaMAX™, penicillin/streptomycin (100 U/mL/100 μg/mL), PHA (3 μg/mL), 10% (fetal bovine serum) FBS, 10 mM 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES) and heparin (125 U.S.P.-U/mL).

All incubations will be done at 37 °C with 5.5 % CO2 in humidified air.
Cytokinesis block (if used):
Cytochalasin B (4 - 6 μg/mL)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and beta-naphthoflavone.
- Species: Rat
- Tissue: Liver
- Inducing Agents: Phenobarbital and beta-naphthoflavone
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes
Test concentrations with justification for top dose:
Experiment I:
4 h exposure (+/- S9): 1633, 2857, 5000 µg/mL
Experiment II:
40 h exposure (- S9): 1633, 2857, 5000 µg/mL
No cytotoxicity was observed up to the highest applied concentration, in the absence and presence of S9 mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water (10% (v/v))
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: demicolcine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Test material added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 h
- Exposure duration/duration of treatment: 3 h (3 h exposure experiments) and 20 h (continous exposure experiments)
- Harvest time after the end of treatment (sampling/recovery times): 16 h in 3 h exposure experiments

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Cytochalasin B (cytB), 4 - 6 µL/mL, added after 16 h recovery periode in 3 h exposure experiment (experiment I) and after 20 h exposure period in continous exposure experiment (experiment II).
- Methods of slide preparation and staining technique used including the stain used:
The lymphocyte cultures were centrifuged and the supernatant was removed. The cells were resuspended in hypotonic solution and fresh methanol/acetic acid fixative was added. The fixative was changed several times by centrifugation and resuspension. A few drops of the cell suspension were transfered to glass slides which were allowed to air dry before staining with Giemsa.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 1000 binucleated cells in duplicate cell cultures (i.e. a minimum of 2000 binucleated cells)
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Only cells containing a clearly visible cytoplasm will be included in the analysis. The micronucleus has to be stained in the same way as the main nucleus and the area of the micronucleus should not extend the third part of the area of the main nucleus. The micronucleus frequency will be reported as % micronucleated cells.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)
Evaluation criteria:
ACCEPTANCE CRITERIA
The assay is considered valid if the following criteria are met:
- The concurrent solvent control is within the laboratory historical solvent control data range (95% confidence interval).
- The concurrent positive controls produce a statistically significant increase in the micronucleus frequency compared with the concurrent solvent control and are within the laboratory historical positive control data range.
- Cell proliferation criteria in the solvent control are considered to be acceptable.
- The appropriate number of doses and cells is analysed.
- The quality of the slides allows the evaluation of an adequate number of cells and concentrations.

EVALUATION CRITERIA
A test item is considered to be clearly negative if, under all experimental conditions considered:
- None of the test item concentrations exhibits a statistically significant increase of the micronucleus frequency compared with the concurrent solvent control.
- There is no concentration-related increase of the micronucleus frequency.
- The results in all evaluated test item concentrations are within the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval)

A test item is considered to be clearly positive if, under all experimental conditions considered:
- At least one of the test item concentrations exhibits a statistically significant increase of the micronucleus frequency compared with the concurrent solvent control.
- The increase of the micronucleus frequency is concentration-related in at least one experimental condition.
- The results are outside the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval).
Statistics:
Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script. A linear regression was performed using a validated test script to assess a possible dose dependency in the rates of micronucleated cells. A trend was judged as significant whenever the p-value (probability value) was below 0.05. Biological and statistical significance were considered together.
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Table 1: Summary of results of the in vitro micronucleus test in human lymphocytes

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

 

interval

concentration

index

in %*

cells

95% Ctrl limit

 

 

in µg/mL

CBPI

 

in %**

in %

Exposure period 4 h without S9 mix

I

40 h

Solvent control1

1.87

 

0.40

0.00 – 1.04

 

 

Positive control2

1.67

23.2

 13.30S

 

 

 

1633

1.79

9.3

0.75

 

 

 

2857

1.86

2.2

0.65

 

 

 

5000

1.85

2.7

0.75

 

Trend test: p-value 0.251

Exposure period 20 h without S9 mix

II

40 h

Solvent control1

1.76

 

0.70

0.00 – 0.86

 

 

Positive control3

1.46

38.9

 3.00S

 

 

 

1633

1.77

n.c.

0.75

 

 

 

2857

1.67

12.0

0.45

 

 

 

5000

1.62

17.6

0.50

 

Trend test: p-value 0.265

Exposure period 4 h with S9 mix

I

40 h

Solvent control1

1.71

 

0.25

0.00 – 1.03

 

 

Positive control4

1.43

39.2

 3.15S

 

 

 

1633

1.69

3.2

0.35

 

 

 

2857

1.76

n.c.

0.50

 

 

 

5000

1.67

5.2

0.60

 

Trend test: p-value 0.016T

*: For the positive control groups and the test item treatment groups the values are related to the solvent controls

**: The number of micronucleated cells was determined in a sample of 2000 binucleated cells

S: The number of micronucleated cells is statistically significantly higher than corresponding control values

T: Trend analysis via linear regression is significant (p ˂ 0.05)

n.c.: Not calculated as the CBPI is equal or higher than the solvent control value

1: Deionised water 10.0 % (v/v)

2: MMC 0.8 µg/mL

3: Demecolcine 125 ng/mL

4: CPA 15.0 µg/mL

Conclusions:
Preliminary interpretation of results: negative and without metabolic activation

No final results can currently be reported. Pursuant to ECHA Decision CCH-D-2114465595-37-01/F (issued 4 July 2019) an in vitro micronucleus study according to OECD guideline 487 is currently being performed. At the time of submission, the experimental phase of the study was completed and preliminary results were available. A written confirmation of the test facility is attached under 'Attached full study report'. A further dossier update will be submitted containing the final results once the final study report is available. Based on the final results, the hazard assessment of the registered substance with respect to the genetic toxicity endpoint cytogenicity / induction of micronuclei formation will be updated.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Mar - 23 Apr 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
adopted in 2016
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted in 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
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:
Batch No.: P2D4000629
Expiry date: 2020-07-04
Appearance: Yellowish Liquid
Storage conditions:Ambient (15 – 25 °C)
Purity: 100% (UVCB as produced)
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHO-K1 cells, obtained from the European Collection of Cell Cultures.
- Suitability of cells: Cell type selected is listed as one of the recommended cell types in OECD guideline 476.

For cell lines:
- Absence of Mycoplasma contamination: checked periodically

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: All cell cultures were maintained at 34 - 39 °C in a humidified atmosphere of 5% CO2 in air.

- H0 medium: Ham’s Nutrient Mixture F12, supplemented with 1 mM L-glutamine and 50 ng/mL amphotericin B / 20 IU/mL penicillin / 20 μg/mL streptomycin
- H10 medium: H0 medium supplemented with 10% heat-inactivated fetal calf serum (HiFCS)
- 6-TG / selective medium: H10 medium supplemented with 6-thioguanine (6-TG) at a final concentration of 10 μg/mL
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
- Species: Rat
- Strain: Sprague Dawley
- Sex: male
- Tissue: Liver
- Inducing Agents: Phenobarbital and 5,6-Benzoflavone
- Producer: MolTox Inc.
Test concentrations with justification for top dose:
Preliminary toxicity test (+/- S9): 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL
Mutation assay (3 h, +/-S9): 312.5, 538, 1250, 2500 and 5000 µg/mL
The selection of the concentrations used in the main experiments was based on data from the preliminary toxicity test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water (10% (v/v))
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : single

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 2 x 10E6
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: approx. 24 h
- Exposure duration/duration of treatment: 3 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): approx. 7 days
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine): 6-thioguanine (6-TG), 10 µg/mL
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 200 (in non-selective medium), 5 × 10E5 (in selective medium)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: Mutant frequency (MF) per million surviving cells
Evaluation criteria:
ACCEPTANCE CRITERIA
Test item:
The highest concentration tested allowed a maximum exposure up to 5000 µg/mL for soluble compounds, or the limit of toxicity (relative survival (RS) reduced to 10 - 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should be achieved which ideally spanned the toxicity range of 10 - 100% RS.
Vehicle control:
- The mean vehicle control value for mutant frequency (MF) was between 1 - 20 x 10E-6.
- The mean cloning efficiency was between 65 - 120%.
- Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining.
- The concurrent vehicle control must be considered acceptable for addition to the laboratories historical vehicle control data base (ideally within the 95% confidence limits).
Positive control:
- Positive controls showed a statistically significant increase in mean total MF above the mean concurrent vehicle control MF and within, or close to, the range of the historical control data.

EVALUATION CRITERIA
A test item is considered to be positive if:
- at least one of the test concentrations exhibits a statistically significant increase in mean mutant frequency compared with the concurrent negative control
- the increase in mean mutant frequency is concentration-related when evaluated with an appropriate trend test
- any of the results (mean mutant frequency) are outside the distribution of the historical negative control data (above the upper 95% confidence limit)
A test item is considered to be negative if:
- none of the test concentrations exhibits a statistically significant increase in mean mutant frequency compared with the concurrent negative control
- there is no concentration-related increase in mean mutant frequency when evaluated with an appropriate trend test
- all results (mean mutant frequency) are inside the distribution of the historical negative control data (within the 95% confidence limits).
Statistics:
The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution. Tests were conducted for a linear concentration-response relationship of the test item, for non-linearity, and for the comparison of positive control and treated groups to solvent control.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Treatment with the test item did not change the pH significantly at any dose level in any experiment.
- Data on osmolality: Treatment with the test item did not increases the osmolality significantly at any dose level in any experiment.
- Precipitation and time of the determination: No precipitation of the test item was noted at any dose level.

RANGE-FINDING/SCREENING STUDIES
A preliminary cytotoxicity assay was performed using a single culture at each test point both in the presence and absence of metabolic activation. No positive controls were included. The test item was assayed at concentrations of 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL. No significant cytotoxic effects were observed. Based on these findings the test item concentrations in the main assay were selected.

STUDY RESULTS
- Concurrent vehicle negative and positive control data are reported in tabular form under 'Any other information on results incl. tables'.

HISTORICAL CONTROL DATA
- Data are presented in tabular form under 'Any other information on results incl. tables'.

Table 1: Summary of results

Test Item

Concentration

(µg/mL)

3-hour Treatment ‑S9 mix

95% confidence limits of HCD

3-hour
Treatment +S9 mix

95% confidence limits of HCD

Mean RS (%)

Mean MFa

Mean RS (%)

Mean MFa

Deionized Water

0

100

7.20

2.4 – 15.3

100

7.95

0.2 – 16.1

Propylidynetrimethanol, propoxylated

312.5

89

8.11

 

92

7.60

 

Propylidynetrimethanol, propoxylated

535[DA1] b

100

6.34

 

79

8.13

 

Propylidynetrimethanol, propoxylated

1250

95

6.84

 

86

9.57

 

Propylidynetrimethanol, propoxylated

2500

92

6.99

 

83

8.93

 

Propylidynetrimethanol, propoxylated

5000

96

9.34

 

55

5.82

 

Ethyl methanesulphonate

250

108

80.71***

42.8 – 131.1

NT

NT

 

3-methylcholanthrene

5

NT

NT

 

103

71.03***

18.7 – 105.5

a. Mutant frequencies expressed per 10E6 viable cells

b. actual concentration

RS: Relative Survival

MF: Mutant Frequency

NT: Not tested

HCD: Historical control data

*** p<0.001; all other cultures p≥0.05.  Treated groups were compared to the vehicle control using one-tailed Dunnett’s tests for an increase and the positive control was compared to the vehicle control using a one-tailed t test for an increase

Table 2: Main Test: 3-hour treatment in the absence of S9 mix, Day 1 relative survival

Concn.of Test item   (µg/mL)

Cell Count Day 1 (x106/mL)

No. of colonies on plate

Total no. of Colonies

Cloning Efficiency (%)

Adjusted Cloning Efficiency

(%)

RS

(%)

Mean RS

(%)

Plate 1

Plate 2

Plate 3

 

 

0a

 

1.15

121

129

134

384

65

69

100

100

1.25

129

133

124

386

 

 

 

 

1.25

124

128

131

383

 

 

 

 

1.25

146

131

125

402

 

 

 

 

 

 

 

 

 

 

 

 

 

 

312.5

1.30

118

132

111

361

60

68

98

89

1.27

110

98

93

301

50

55

80

 

538 b

1.31

143

114

139

396

66

75

109

100

1.31

106

110

114

330

55

62

91

 

1250

1.27

128

91

98

317

53

58

84

95

1.18

163

128

139

430

72

73

106

 

2500

1.16

135

115

144

394

66

66

96

92

1.21

106

107

137

350

58

61

89

 

5000

1.22

123

126

112

361

60

64

92

96

1.23

115

138

133

386

64

68

100

 

  EMS – positive control

 

 

 

 

 

 

 

250

1.10

163

153

151

467

78

74

108

108

1.26

135

143

132

410

68

74

108

 

a. Vehicle control = Deionized Water 10% (v/v)

b. actual concentration

RS: Relative Survival

EMS: Ethyl methanesulphonate

Cell count pre-treatment = 1.2 x 10E6 cells/mL

Test item = Propylidynetrimethanol, propoxylated

Table 3: Main Test: 3-hour treatment in the absence of S9 mix, Day 8 cloning efficiency

Concn.of Test item   (µg/mL)

No. of colonies on plate

Total no. of Colonies

Cloning Efficiency in non‑selective medium (%)

Plate 1

Plate 2

Plate 3

 

 

0a

 

146

143

142

431

72

139

141

146

426

71

149

143

147

439

73

149

148

141

438

73

 

 

 

 

 

 

312.5

138

143

145

426

71

142

146

145

433

72

538 b

139

149

153

441

74

143

138

147

428

71

1250

143

146

134

423

71

138

142

140

420

70

2500

129

127

121

377

63

135

120

123

378

63

5000

125

123

120

368

61

142

153

147

442

74

  EMS – positive control

 

 

 

250

126

134

118

378

63

117

121

128

366

61

a. Vehicle control = Deionized Water 10% (v/v)

b. actual concentration

EMS: Ethyl methanesulphonate

Test item = Propylidynetrimethanol, propoxylated

Table 4: Main Test: 3-hour treatment in the absence of S9 mix, mutant frequency

Concn.of Test item   (µg/mL)

No. of colonies on plate

Total no. of Colonies

Cloning Efficiency in selective medium (%)

Mutant Frequencya

Mean Mutant Frequencya

   p‑valuec   

Plate

1

Plate

 2

Plate 3

Plate 4

Plate 5

 

 

0b

 

3

2

2

3

3

13

0.00052

7.24

7.20

 

4

2

1

2

4

13

0.00052

7.32

 

 

1

2

0

3

3

9

0.00036

4.92

 

 

4

2

2

4

5

17

0.00068

9.32

 

 

 

 

 

 

 

 

 

 

 

 

 

312.5

3

3

1

7

3

17

0.00068

9.58

8.11

0.697

2

4

3

0

3

12

0.00048

6.65

 

 

538d

2

2

2

4

3

13

0.00052

7.07

6.34

0.951

1

2

1

3

3

10

0.00040

5.61

 

 

1250

0

2

3

3

2

10

0.00040

5.67

6.84

0.916

5

2

2

3

2

14

0.00056

8.00

 

 

2500

2

0

1

3

1

7

0.00028

4.46

6.99

0.963

2

0

1

7

5

15

0.00060

9.52

 

 

5000

1

5

2

0

4

12

0.00048

7.83

9.34

0.390

4

3

3

6

4

20

0.00080

10.86

 

 

  EMS – positive control

 

 

 

 

 

 

 

 

250

16

24

29

27

25

121

0.00484

76.83

80.71

<0.001***

30

26

27

18

28

129

0.00516

84.59

 

 

a. Mutant frequencies expressed per 10E6 viable cells

b. Vehicle control = Deionized Water 10% (v/v)

c. p-values are for comparison of treated groups to vehicle control using one-tailed Dunnett’s test and comparison of positive control to the vehicle control using a one-tailed t test

d. actual concentration

*** p<0.001; all other cultures p≥0.05

EMS: Ethyl methanesulphonate

Test item = Propylidynetrimethanol, propoxylated

Table 5: Main Test: 3-hour treatment in the presence of S9 mix, Day 1 relative survival

Concn.of Test item   (µg/mL)

Cell Count Day 1 (x106/mL)

No. of colonies on plate

Total no. of Colonies

Cloning Efficiency (%)

Adjusted Cloning Efficiency

(%)

RS

(%)

Mean RS

 (%)

Plate 1

Plate 2

Plate 3

 

 

0a

 

1.19

143

143

151

437

75

66

100

100

1.04

157

140

157

454

 

 

 

 

1.17

141

162

154

457

 

 

 

 

1.03

156

142

161

459

 

 

 

 

 

 

 

 

 

 

 

 

 

 

312.5

1.23

130

133

110

373

62

61

92

92

1.15

127

137

132

396

66

60

92

 

538b

1.24

102

96

80

278

46

45

69

79

1.13

130

141

118

389

65

58

88

 

1250

1.09

122

174

165

461

77

66

101

86

1.14

97

112

99

308

51

46

71

 

2500

1.11

160

128

148

436

73

64

97

83

1.14

95

99

110

304

51

45

69

 

5000

1.02

93

86

76

255

43

34

52

55

0.97

97

119

81

297

50

38

58

 

  3MC – positive control

 

 

 

 

 

 

 

5

1.16

152

140

153

445

74

68

103

103

1.06

160

164

160

484

81

68

103

 

a. Vehicle control = Deionized Water 10% (v/v)       

b. actual concentration

RS: Relative Survival

3MC: 3-Methylcholanthrene

Cell count pre-treatment = 1.3 x 10E6 cells/mL

Test item = Propylidynetrimethanol, propoxylated

Table 6: Main Test: 3-hour treatment in the presence of S9 mix, Day 8 cloning efficiency

Concn.of Test item   (µg/mL)

No. of colonies on plate

Total no. of Colonies

Cloning Efficiency in non‑selective medium (%)

Plate 1

Plate 2

Plate 3

 

 

0a

 

142

141

138

421

70

133

143

146

422

70

135

147

152

434

72

136

139

140

415

69

 

 

 

 

 

 

312.5

135

136

135

406

68

138

137

142

417

70

538 b

135

135

148

418

70

132

137

141

410

68

1250

145

151

142

438

73

145

152

143

440

73

2500

142

135

141

418

70

139

135

142

416

69

5000

109

117

121

347

58

121

111

122

354

59

3MC – positive control

 

 

 

5

115

117

124

356

59

123

124

117

364

61

a. Vehicle control = Deionized Water 10% (v/v)

b. actual concentration

3MC: 3-Methylcholanthrene

Test item = Propylidynetrimethanol, propoxylated

Table 7: Main Test: 3-hour treatment in the presence of S9 mix, mutant frequency

Concn.of Test item   (µg/mL)

No. of colonies on plate

Total no. of Colonies

Cloning Efficiency in selective medium (%)

Mutant Frequencya

Mean Mutant Frequencya

   p‑valuec   

Plate
 1

Plate

2

Plate 3

Plate 4

Plate 5

 

 

0b

 

3

1

3

3

2

12

0.00048

6.84

7.95

 

2

1

4

5

3

15

0.00060

8.53

 

 

3

2

1

1

6

13

0.00052

7.19

 

 

3

3

4

2

4

16

0.00064

9.25

 

 

 

 

 

 

 

 

 

 

 

 

 

312.5

2

4

2

4

3

15

0.00060

8.87

7.60

0.939

4

2

2

3

0

11

0.00044

6.33

 

 

538 d

3

2

1

3

2

11

0.00044

6.32

8.13

0.903

4

3

2

6

2

17

0.00068

9.95

 

 

1250

4

2

2

2

6

16

0.00064

8.77

9.57

0.523

2

4

8

2

3

19

0.00076

10.36

 

 

2500

1

2

2

1

4

10

0.00040

5.74

8.93

0.896

5

7

3

4

2

21

0.00084

12.12

 

 

5000

3

0

2

1

2

8

0.00032

5.53

5.82

0.995

0

2

0

6

1

9

0.00036

6.10

 

 

  3MC – positive control

 

 

 

 

 

 

 

 

5

24

25

20

22

18

109

0.00436

73.48

71.03

<0.001***

16

25

20

26

17

104

0.00416

68.57

 

 

a. Mutant frequencies expressed per 106 viable cells

b. Vehicle control = Deionized Water 10% (v/v)

c. p-values are for comparison of treated groups to vehicle control using one-tailed Dunnett’s test and comparison of positive control to the vehicle control using a one-tailed t test

d. actual concentration

*** p<0.001; all other cultures p≥0.05

3MC: 3-Methylcholanthrene

Test item = Propylidynetrimethanol, propoxylated

Table 8: Historical control data in the absence of S9 mix

Data collection period: 06 May 2018 to 03 April 2020

Positive control: Ethyl methanesulphonate (250 µg/mL)

Mean mutant frequency (10-6)

Vehicle control

Positive control

Minimum

4.3

33.3

Maximum

14.6

124.9

Mean

8.9

86.9

Standard Deviation

3.2

22.1

Lower 95% Confidence Limit

2.4

42.8

Upper 95% Confidence Limit

15.3

131.1

Number of tests

26

 

 

Table 9: Historical control data in the presence of S9 mix

Data collection period: 16 April 2018 to 03 April 2020

Positive control: 3-methylcholanthrene (5 µg/mL)

Mean mutant frequency (10-6)

Vehicle control

Positive control

Minimum

2.4

22.1

Maximum

17.6

132.1

Mean

8.2

62.1

Standard Deviation

4.0

21.7

Lower 95% Confidence Limit

0.2

18.7

 Upper 95% Confidence Limit

16.1

105.5

Number of tests

32

 

 

Conclusions:
Interpretation of results: negative with and without metabolic activation
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in bacteria

In an in vitro assessment of the mutagenic potential of the registered substance in bacteria according to OECD guideline 471 under GLP conditions (rel 1-key, AMES, OECD 471, Huntingdon, 2008, PGF0009), histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test substance diluted in water. Water was also used as a negative control. Two independent experiments were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test substance up to 5000 μg/plate were tested. No signs of toxicity were observed towards the tester strains in either mutation test. No evidence of mutagenic activity was seen at any concentration of the test substance in either mutation test, in the absence or presence of metabolic activation. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory. It is concluded that propylidentrimethanol, propoxylated showed no evidence of mutagenic activity in this bacterial system under the test conditions used.

The findings of the key study are supported by the results of a further study investigating gene mutation in Salmonella typhimurium strains TA1535, 1537,  98 and 100 (rel 2, AMES,  OECD 471, Bayer, 1985, T 1019632, PH-13676). The study did not observe GLP conditions and only four tester strains were used. No evidence of mutagenic activity of the test substace was found, with or without metabolic activation. There was neither a dose-related doubling or a biologically relevant increase in the mutant count when compared with the negative controls. All positive controls showed a marked mutagenic effect.

Cytogenicity / micronucleus study

Preliminary result: not genotoxic in human lymphocytes with and without metabolic activation.

Pursuant to ECHA Decision CCH-D-2114465595-37-01/F (issued 4 July 2019) an in vitro micronucleus study according to OECD guideline 487 is currently being performed. At the time of submission, the experimental phase of the study was completed and preliminary results were available, indicating no genotoxicity in human lymphocytes with and without metabolic activation. A further dossier update will be submitted containing the final results once the final study report is available.

Gene mutation in mammalian cells

Propylidynetrimethanol, propoxylated was tested for mutagenic potential in an in vitro mammalian cell mutation assay according to OECD guideline 476 under GLP conditions using Chinese hamster ovary (CHO-K1) cells (rel 1-key, HPRT, OECD 476, Covance, 2020, JJ98HF). Two independent experiments, one in the absence of metabolic activation (S9 mix) and one in the presence of S9 mix, were performed. The vehicle for the test item was deionised water. Cells were exposed to the test substance at concentrations from 312.5 to 5000 µg/mL. No precipitate and no cytotoxicity were observed under any experimental condition used. The test substance did not induce a statistically significant increase in mean mutant frequencies. None of the treated groups induced mean mutant frequencies above the laboratory historical control data 95% confidence limits and tests for both a linear trend and non-linearity were applied across all treatment groups, neither of which was statistically significant. The positive controls (ethyl methane sulphonate in the abseence and 3-methylcholanthrene in the presence of S9 mix) induced a significant increase in mean mutant frequencies demonstrating the correct functioning of the assay. It was concluded that propylidynetrimethanol, propoxylated did not demonstrate mutagenic potential in the in vitro HPRT mutation assay, under the experimental conditions used.

Justification for classification or non-classification

The available data on gene mutation in bacteria and gene mutation in mammalian cells with the registered substance do not meet the criteria for classification according to the CLP Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification. Only preliminary data on cytogenicity / micronuclei formation are available. An adequate and reliable experimental investigation is currently being conducted. The hazard assessment and conclusion on the classification of propylidentrimethanol, propoxylated will be updated and the result of the study on cytogenicity / micronuclei formation will be provided once the study report is available.

The data currently available do not provide sufficient evidence that would imply a classification for genetic toxicity according to the CLP Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification. However, as only preliminary information regarding cytogenicity / micronuclei formation are available, the overall conclusion for classification regarding genetic toxicity needs to be re-assessed once reliable final results are available.