Propylidynetrimethanol, propoxylated

Endpoint summary

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 micronucleus study (OECD 487): negative with and without metabolic activation in human lymphocytes

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

Reference

Reference 1

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)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, GLP Monitoring Authority, UK

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; the top dose is the standard limit concentration as recommended in the regulatory testing guidelines.

Vehicle / solvent:

The test substance 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 the test substance 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

True negative controls:

no

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 h. 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 min 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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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 observed following exposure to the test substance. 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 the test substance 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 second assay. No evidence of toxicity was noted following exposure to the test substance. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

Additional data supporting the information is attached below under 'Attached background material'.

Conclusions:

It is concluded that propylidentrimethanol, propoxylated showed no evidence of mutagenic activity in bacteria under the test conditions used.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Apr - 28 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

Test design, recovery phase and harvest time slightly modified to achieve statistical significant responses for the positive controls.

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Expiry date: 2021-06-30
Appearance: Liquid, clear colorless
Storage conditions: Ambient (15 – 25 °C)
Purity: 99.1% (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, not receiving medication: male (23 years, Experiment I) and female (30 years, Experiment II)
- Whether whole blood or separated lymphocytes were used: Whole blood (11% mixture of whole blood in medium within 30 h after blood collection)
- Whether blood from different donors were pooled or not: Blood was not pooled
- 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 were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

Cytochalasin B (4 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
- S9 composition: MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4)
- Protein concentration: 29.0 mg/mL (Lot no. 050919D)

Test concentrations with justification for top dose:

Experiment I: 4 h exposure (+/- S9): 32.5, 56.8, 99.5, 174, 305, 533, 933, 1633, 2857, 5000 µg/mL
Experiment II: 40 h exposure (- S9): 174, 305, 533, 933, 1633, 2857, 5000 µg/mL
The latter 3 concentrations in each experiment were used for evaluation. 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: 4 h (pulse exposure experiments) and 20 h (continous exposure experiments)
- Harvest time after the end of treatment (sampling/recovery times): 16 h in 4 h exposure experiments

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Cytochalasin B (cytB), 4 µL/mL, added after 16 h recovery periode in 4 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 were included in the analysis. The micronucleus had 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 was 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 in micronucleus frequency compared with the concurrent solvent control.
- There is no concentration-related increase in 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 in micronucleus frequency compared with the concurrent solvent control.
- The increase in 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 (p < 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 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

Remarks:

5000 µg/mL

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: No relevant influence on pH was observed at the highest tested concentration of the test item.
- Data on osmolality: No relevant influence on osmolarity was observed at the highest tested concentration of the test item.
- Possibility of evaporation from medium: The vapour pressure of the test subsance is 0.00179 Pa at 20°C. Hence, evaporation from the dose formulations is not considered to be significant.
- Water solubility: The water solubility was 100 g/L at 20 °C.
- Precipitation and time of the determination: No precipitation occurred at any time in any experiment.
- Definition of acceptable cells for analysis: Refer to 'Details on test system and experimental conditions'.

RANGE-FINDING/SCREENING STUDIES:
Since the test substance is of UVCB nature, 5000 µg/mL was selected as the top concentration, according to the provision of OECD guideline 487, adopted July 2016. Test item concentrations ranging from 32.5 to 5000 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In this study in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated as Experiment I.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Refer to attached pdf documents under 'Attached background material'.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : Refer to attached pdf documents under 'Attached background material'.
- Statistical analysis; p-value: Refer to attached pdf documents under 'Attached background material'.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: Refer to attached pdf documents under 'Attached background material'.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Refer to attached pdf documents under 'Attached background material'.
- Negative (solvent/vehicle) historical control data: Refer to attached pdf documents under 'Attached background material'.

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 control^1	1.87		0.40	0.00 – 1.04
		Positive control^2	1.67	23.2	13.30^S
		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 control^1	1.76		0.70	0.00 – 0.86
		Positive control^3	1.46	38.9	3.00^S
		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 control^1	1.71		0.25	0.00 – 1.03
		Positive control^4	1.43	39.2	3.15^S
		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 0.016^T

*: 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

For concentrations applied, detailed information on results, data on cytotoxicity, number of micronucleated cells, details on biometry and historical control data, please refer to the attached pdf documents under 'Attached background material'.

Conclusions:

Interpretation of results: negative with and without metabolic activation

Reference 3

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

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

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted in 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

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:

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 MF^a		Mean RS (%)	Mean MF^a
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

Conc^n.of Test item   (µg/mL)	Cell Count Day 1 (x10^6/mL)	No. of colonies on plate			Total no. of Colonies	Cloning Efficiency (%)	Adjusted Cloning Efficiency (%)	RS (%)	Mean RS (%)
		Plate 1	Plate 2	Plate 3
0^a	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

Conc^n.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
0^a	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

Conc^n.of Test item   (µg/mL)	No. of colonies on plate					Total no. of Colonies	Cloning Efficiency in selective medium (%)	Mutant Frequency^a	Mean Mutant Frequency^a	p‑value^c
	Plate 1	Plate  2	Plate 3	Plate 4	Plate 5
0^b	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
538^d	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

Conc^n.of Test item   (µg/mL)	Cell Count Day 1 (x10^6/mL)	No. of colonies on plate			Total no. of Colonies	Cloning Efficiency (%)	Adjusted Cloning Efficiency (%)	RS (%)	Mean RS  (%)
		Plate 1	Plate 2	Plate 3
0^a	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
538^b	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

Conc^n.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
0^a	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

Conc^n.of Test item   (µg/mL)	No. of colonies on plate					Total no. of Colonies	Cloning Efficiency in selective medium (%)	Mutant Frequency^a	Mean Mutant Frequency^a	p‑value^c
	Plate  1	Plate 2	Plate 3	Plate 4	Plate 5
0^b	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 vitro

In an in vitro assessment of the mutagenic potential of propylidentrimethanol, propoxylated (CAS No. 25723-16-4, EC No. 500-041-9) 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.

Micronucleus study in vitro

The potential of propylidentrimethanol, propoxylated (CAS No. 25723-16-4, EC No. 500-041-9) to induce micronuclei in human lymphocytes in vitro was investigated in two independent experiments performed according to OECD guideline 487 under GLP conditions (rel-1, MNT, OECD 487, ICCR, 2020, 1952006). In each experimental group, two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. Since the test substance is of UVCB nature, the highest concentration applied  was 5000 μg/mL in accordance with the provision of the current OECD guideline. The following concentrations were used: Experiment I (4 h exposure, +/- S9): 32.5, 56.8, 99.5, 174, 305, 533, 933, 1633, 2857, 5000 µg/mL; Experiment II (40 h exposure, - S9): 174, 305, 533, 933, 1633, 2857, 5000 µg/mL. Deionised water was used as vehicle. The latter 3 concentrations in each experiment were used for the evaluation of micronuclei formation. No cytotoxicity was observed up to the highest applied concentration, in the absence and presence of S9 mix. In Experiment I and II in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test subsstance. In the presence of S9 mix, however, dose dependency, tested by trend test was observed. However, since none of the values were statistically significantly increased and all values were clearly within the historical control data range, this finding was considered biologically irrelevant. Appropriate mutagens were used as positive controls and induced statistically significant increases in cells with micronuclei, as expected. In conclusion, it can be stated that under the experimental conditions reported, propylidynetrimethanol, propoxylated did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes, when tested up to the highest required concentration. Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test.

Gene mutation in mammalian cells in vitro

Propylidynetrimethanol, propoxylated (CAS No. 25723-16-4, EC No. 500-041-9) 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, micronucleus formation in mammalian cells and gene mutation in mammalian cells with propylidentrimethanol, propoxylated (CAS No. 25723-16-4, EC No. 500-041-9) are all negative. They 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.