Isooctadecanoic acid, monoester with propane-1,2-diol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471, read across): negative with and without metabolic activation in S. typhimurium TA 1535, 1537, 1538, 98 and 100, and 102

Gene mutation in mammalian cells (OECD 490): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Chromosome aberration (OECD 473): negative in primary human peripheral lymphocytes with and without metabolic activation

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating 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:

Data from the source substance Octadecanoic acid, monoester with 1,2-propanediol / Palmitic acid, monoester with 1,2-propanediol (CAS 1323-39-3 / 29013-28-3) was selected as key results for reasons of structural similarity and data reliability.
Additional bacterial mutagenicity data were available for the source substances Stearic acid, monoester with propane-1,2-diol (CAS 1323-39-3) and Fatty acids, C14-18 and C16-18-unsatd., esters with propylene glycol (CAS 84988-75-0). Both substances were tested in a bacterial reverse mutation study (Ames test) using three or five S. typhimurium strains. No mutagenicity in bacteria was seen for both source substance under the given test conditions.

Remarks on result:

other: Source: CAS 1323-39-3/29013-28-3

Conclusions:

The read across approach is justified in the analogue justification. The target and source substances are considered unlikely to differ in their genetic toxicity potential. In three bacterial reverse mutation assays (Ames Test) with the three source substances a) stearic acid, monoester with propane-1,2-diol / 2-hydroxypropyl stearate (CAS 1323-39-3)/Palmitic acid, monoester with 1,2-propanediol (CAS 29013-28-3), b) stearic acid, monoester with propane-1,2-diol / 2-hydroxypropyl stearate (CAS 1323-39-3), and c) Fatty acids, C14-18 and C16-18-unsatd., esters with propylene glycol (CAS 85988-75-0) no mutagenicity in bacteria was found. Therefore no mutagenicity in bacteria is expected for target substance isooctadecanoic acid, monoester with propane-1,2-diol (CAS 68171-38-0).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Aug - 08 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted in 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Medicine & Healthcare products Regulatory Agency, United Kingdom

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK (Cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978).
- Cell generation time: approximately 12 hours

MEDIA USED
- Type and identity of media including carbon dioxide concentration if applicable: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at approximately 37 degree Celsius with 5% carbon dioxide in air.
- Properly maintained: yes; the cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasm
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes, The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

other:

Remarks:

L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus)

Metabolic activation:

with and without

Metabolic activation system:

2% S9; Lot No. PB/βNF S9 30/06/17. Obtained from Sprague-Dawley rats after induction with phenobarbital / beta-naphtha flavone at 80/100 mg/kg bw. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in an Ames test.

Test concentrations with justification for top dose:

4-hour without S9: 10, 20, 40, 50, 60 µg/mL
4-hour with S9 (2%): 20, 40, 50, 60, 70 µg/mL
24-hour without S9: 20, 40, 50, 60, 70, 80 µg/mL
The dose levels were limited by test item induced toxicity

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Fisher batches 1684307 Expiry 03/10/22 purity > 99%)
- Justification for choice of solvent/vehicle: test item not soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium in tissue culture flasks
- Cell density at seeding (if applicable): 1 x 10e6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10e6 cells/mL in 10 mL cultures were established in 25 cm² tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation.
The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).
2 mL of S9-mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group) were added to each universal.

DURATION
- Exposure duration: 4 or 24 hours

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS:
The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at 8 dose levels of the test item, vehicle and positive controls.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG) and percentage relative suspension growth (%RSG)
- Any supplementary information relevant to cytotoxicity: The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Measurement of Survival, Viability and Mutant Frequency
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10e5 cells/mL. The cultures were incubated at 37°C with 5% carbon dioxide in air and subcultured every 24 hours for the expression period of two days, by counting and diluting to 2 x 10e5 cells/mL, unless the mean cell count was less than 3 x 10e5 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 10e4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37°C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al, 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Percentage Relative Suspension Growth (%RSG), Day 2 Viability (%V), and Mutation Frequency (MF) were calculated.

Evaluation criteria:

-The majority of plates, for both viability (%V) and TFT resistance, are analysable for each experiment.
-The absolute viability (%V) at the time of mutant selection of the solvent controls is 65 to 120%.
-The total suspension growth of the solvent control following 4-hour exposure, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24-hour exposure the total suspension growth should be in the range of 32 to 180.
-The in-house vehicle control mutant frequency is in the range of 50–170x10e-6 cells. Experiments where the vehicle control values are markedly greater than 200x10e-6 mutant frequency per survivor are not acceptable.
-The positive control should demonstrate an absolute increase in total MF, that is, an increase above the spontaneous background MF of at least 300x10e-6. At least 40% of the IMF should be reflected in the small colony MF.
The positive control has an increase in the small colony MF of at least 150x10e-6 above that seen in the concurrent untreated/solvent control (a small colony IMF of 150x10e-6).
- The upper limit of cytotoxicity observed in the positive control should be the same as for the experimental cultures i.e. the Relative Total Growth (RTG) and percentage Relative Suspension Growth (%RSG) should be greater than approximately 10 % of the concurrent selective control group.
- For non-toxic test items the upper test item concentrations are 10 mM, 2 mg/mL or 2 μL/mL whichever is lowest. When the test item is a substance of unknown or variable composition (UVCBs) the upper dose level may need to be higher and the maximum concentration is 5 mg/mL. Precipitating dose levels are not tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point. In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20% of survival.

Statistics:

Descriptive statistics

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The maximum dose level used induced toxicity.

Vehicle controls validity:

valid

Remarks:

The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus.

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels were limited by test item induced toxicity. No precipitate of the test item was observed at the end of exposure.

HISTORICAL CONTROL DATA
Given with ranges, means and standard deviation (no confidence interval)
- Positive historical control data: given for 20 experiments with and without metabolic activation
- Negative (solvent/vehicle) historical control data: given for solvent for 20 experiments with and without metabolic activation

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: %RSG (relative suspension growth)

Remarks on result:

other: Mutant frequencies were within the range of historical vehicle and positive controls

The substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor of 126 x 10 e-6, consequently it is considered to be non-mutagenic in this assay. The Global Evaluation Factor is a predefined induced mutant frequency (i.e. increase in mutant frequency above the concurrent control) based on the analysis of the distribution of the vehicle control mutant frequency data from participating laboratories.

Preliminary cytotoxicity test

Dose (μg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
0.61	86	109	70
1.22	95	112	79
2.44	93	104	92
4.88	106	109	80
9.77	96	107	98
19.53	99	115	73
39.06	118	108	72
78.13	9	35	9
156.25	0 p	0 p	0 p

p = precipitate; RSG = relative suspension growth

 

Main Experiment (4 -hours-S9)

Treatment (μg/mL)	%RSG	RTG	MF§
0	100	1.00	150.31
10	97	0.99	145.17
20	98	0.93	139.58
40	105	0.95	134.41
50	66	0.63	133.41
60	9	0.10	195.26
Ethylmethanesulphonate 400	80	0.62	985.92
Historical vehicle control			147.37, standard deviation 24.21
Historical positive control			1359.98, standard deviation 408.24

 

 Main Experiment (4 -hours+S9)

Treatment (μg/mL)	%RSG	RTG	MF§
0	100	1.00	122.77
20	88	0.88	130.66
40	87	0.83	138.73
50	88	0.85	131.35
60	61	0.62	134.14
70	23	0.24	106.88
Cyclophosphamide 1.5	82	0.83	496.22
Historical vehicle control			136.31 , Standard deviation 15.75
Historical positive control			1013.36, standard deviation 226.01

 

Main Experiment (24 -hours-S9)

Treatment (μg/mL)	%RSG	RTG	MF§
0	100	1.00	146.71
20	91	1.06	139.18
40	83	0.99	155.49
50	77	0.94	161.45
60	63	0.84	145.06
70	33	0.60	145.89
80	12	0.30	157.37
Ethylmethanesulphonate 150	41	0.34	2087.13
Historical vehicle control			147.37, standard deviation 24.2
Historical positive control			1359.98, standard deviation 408.24

§ = Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis

%RSG = Relative Suspension Growth; RTG = Relative Total Growth

MF§ = 5-TFT resistant mutants/10e6 viable cells 2 days after treatment

The Historical Vehicle and Positive Control data was generated by a program on a rolling system of the last twenty sets of archived data. The program combines the 4 -hour and 24 -hour data in the absence of metabolic activation as the acceptability criteria is the same for all of the exposure groups.

Conclusions:

Interpretation of results: negative.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Aug -19 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE)

Version / remarks:

Guidelines of 31 March 2011

GLP compliance:

yes (incl. QA statement)

Remarks:

Medicine & Healthcare products Regulatory Agency, United Kingdom

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

primary culture, other: human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: female donor
- Suitability of cells: Lymphocytes (whole blood cultures)
- Cell cycle length, doubling time or proliferation index: average generation time (AGT) approximately 16 hours (laboratory's experience)
- Sex, age and number of blood donors if applicable: one female donor, 26 years for preliminary toxicity test, one female donor, 28 years for main experiment
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including carbon dioxide concentration if applicable: Eagle's MEM with HEPES buffer, supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 ºC with 5% carbon dioxide
- Properly maintained: yes

The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Metabolic activation:

with and without

Metabolic activation system:

2% S9-mix (Microsomal fractions, standardized laboratory's preparation, Sprague-Dawley rats treated with phenobarbital/beta-naphtha flavone at 80/100 mg/kg bw)

Test concentrations with justification for top dose:

0, 20, 40, 60, 80, 100, 120, 160 µg/mL
The maximum dose level tested in the main test was the lowest precipitating dose level; the dose range also included a dose level that induced greater than optimum toxicity with approximately 55±5% mitotic inhibition.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in aqueous culture media at 50 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 500 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide (DMSO), batch: 1684307, purity >99%

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

CULTURE CONDITIONS:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 9.05 mL MEM with 10% (FBS), 0.1 mL Li-heparin, 0.1 mL phytohaemagglutinin, 0.75 mL heparinized whole blood

DURATION
- Preincubation period: 48h
- Exposure durations: 4h with S9, 4h without S9 , and 24h without S9
- Fixation time (start of exposure up to harvest of cells): 4h exposure: 24h; 24 exposure: 24h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 μg/mL, 2.5h before harvest time

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: duplicates

METHODS OF SLIDE PREPARATION:
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;
- Any supplementary information relevant to cytotoxicity: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

OTHER EXAMINATIONS:
- Determination of polyploidy and endoreplication: Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range was shown in the report.

- OTHER:
Experimental start date: 31 Aug 2017
Experimental completion date: 10 Nov 2017

Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made.

Evaluation criteria:

- The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
- All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
- The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
- The required number of cells and concentrations were analyzed.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps was <0.05 when compared to its concurrent control and there was a dose-related increase in the frequency of cells with aberrations. Incidences where marked statistically significant increases were observed only with gap-type aberrations were assessed on a case by case basis.

Key result

Species / strain:

primary culture, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate observations were made at the end of exposure in blood-free cultures and was noted at 160 μg/mL in all exposure groups.
- Other confounding effects: Haemolysis was also noted at and above 60 μg/mL in the 4(20)-hour exposure group in the absence of S9 and at 160 μg/mL in the presence of S9 and in the 24-hour exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes and was therefore judged not to impact the results.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to the maximum dose level of 5000 μg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). In the 24-hour continuous exposure group metaphases were present up to 156.25 μg/mL and at 2500 μg/mL and 5000 μg/mL. The re-occurrence of metaphases at 2500 μg/mL and 5000 μg/mL is considered to be due to the increasing level of precipitate effectively reducing exposure to the cells. The maximum dose level selected for mitotic index evaluation was the lowest precipitating dose level, 156.25 μg/mL. The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level with toxicity also being taken into account in the dose range selected. The maximum dose level selected was 160 μg/mL for all three exposure groups.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: yes (see Table 6 below). The results were acceptable compared with the ranges given in historical controls.
- Negative vehicle historical control data: yes (see Table 5 below). The results were acceptable compared with the ranges given in historical controls.

Table 1: Results of Chromosome Aberration Test, Main Experiment 4h without S9, total of 2 duplicates

Group	Mitotic Index [%]	Number of cells scored	Total number (%) of aberrations with gaps	Total number (%) of aberrations without gaps
Vehicle control (DMSO)	100	300	2 (0.7%)	2 (0.7%)
40 µg/mL	76	300	1 (0.3%)	0 (0%)
60 µg/mL	75	300	5 (1.7%)	4 (1.3%)
80 µg/mL	60	300	5 (1.7%)	2 (0.7%)
100 µg/mL	32	300	1 (0.3%)	0 (0%)
Mitomycin C 0.4 µg/mL	38	88	53 (60%)	50 (62.5%)

 

Table 2: Results of Chromosome Aberration Test, Main Experiment 4h with S9, total of 2 duplicates

Group	Mitotic Index [%]	Number of cells scored	Total number (%) of aberrations with gaps	Total number (%) of aberrations without gaps
Vehicle control (DMSO)	100	300	1 (0.3%)	1 (0.3%)
60 µg/mL	87	300	3 (1%)	2 (0.7%)
80 µg/mL	88	300	0 (0%)	0 (0%)
100 µg/mL	85	300	3 (1%)	3 (1%)
120 µg/mL	36	300	3 (1%)	3 (1%)
Cyclophosphamide 0.4 µg/mL	79	209	40 (19.1%)	35 (16.7%)

 

Table 3: Results of Chromosome Aberration Test, Main Experiment 24h without S9, total of 2 duplicates

Group	Mitotic Index [%]	Number of cells scored	Total number (%) of aberrations with gaps	Total number (%) of aberrations without gaps
Vehicle control (DMSO)	100	300	0 (0%)	0 (0%)
60 µg/mL	92	300	2 (0.7%)	0 (0%)
80 µg/mL	85	300	0 (0%)	0 (0%)
100 µg/mL	68	300	2 (0.7%)	1 (0.3%)
120 µg/mL	46	300	1 (0.3%9	0 (0%)
Mitomycin C 0.2 µg/mL	27	106	31 (29.2%)	27 (25.5%)

 

Table 4: Mean Frequency of Polyploid Cells (%), Main Experiment

Dose Level (µg/mL)	4(20)-Hour Without S9	4(20)-Hour With S9	24-Hour Without S9
0	0.3	0	0
40	0	-	-
60	0	0	0
80	0	0	0
100	0	0	0
120	-	0.3	0.3
MMC 0.4	0	NA	NA
MMC 0.2	NA	NA	0
CP 2	NA	0	NA

MMC = Mitomycin C; CP = Cyclophosphamide; NA = Not applicable; -= Not assessed for mitotic index

 

Table 5: Historical Aberration Ranges for Vehicle Control Cultures

	4h –S9	4h –S9	4h +S9	4h +S9	24h –S9	24h –S9
	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids
Minimum	0	0	0	0	0	0
Maximum	2.5	1	3	0.5	2.5	0.5
Mean	0.56	0.07	0.43	0.03	0.51	0.03
Standard Deviation	0.60	0.24	0.66	0.12	0.61	0.12
Number	52	52	52	52	52	52

 

Table 6: Historical Aberration Range for Positive Control Cultures

	4h –S9	4h –S9	4h +S9	4h +S9	24h –S9	24h –S9
	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids	% cells with aberrations (-gaps)	% cells with polyploids
Minimum	11	0	12.5	0	20	0
Maximum	58	0	47	0.5	62	1
Mean	37.76	0.00	27.01	0.02	40.67	0.02
Standard Deviation	12.63	0.00	9.46	0.10	11.05	0.14
Number	52	52	52	52	52	52

 

Table 7: Mitotic Index – Main Experiment(4(20)-hour Exposure Groups)

Dose Level (mg/mL)	4h –S9	4h –S9	4h –S9	4h –S9	4h +S9	4h +S9	4h +S9	4h +S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	10.80	10.15	10.48	100	9.60	9.20	9.40	100
20	-	-	-	-	-	-	-	-
40	9.05	6.85	7.95	76	-	-	-	-
60	9.35 H	6.45 H	7.90	75	8.50	7.80	8.15	87
80	6.35 H	6.25 H	6.30	60	8.85	7.70	8.28	88
100	3.60 H	3.00 H	3.30	32	8.35	7.60	7.98	85
120	- H	- H	-	-	2.80	3.90	3.35	36
160	- H P	- H P	-	-	NM H P	NM H P	-	-
MMC 0.4	3.75	4.20	3.98	38	NA	NA	NA	NA
CP 2	NA	NA	NA	NA	7.80	7.00	7.40	79

MMC = Mitomycin C; CP = Cyclophosphamide; P = Precipitate; NA = Not applicable; -= Not assessed for mitotic index; NM = No metaphases suitable for scoring; H = Hemolysis

 

Table 8: Mitotic Index – Main Experiment (24-hour Exposure Group)

Dose Level (µg/mL)	A	B	Mean	% of Control
0	7.00	5.35	6.18	100
20	-	-	-	-
40	-	-	-	-
60	6.20	5.20	5.70	92
80	4.50	6.00	5.25	85
100	3.95	4.50	4.23	68
120	3.33	2.30	2.82	46
160	NM H P	NM H P	NM	NM
MMC 0.2	1.50	1.85	1.68	27

 

Conclusions:

Interpretation of results: negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.

 

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS1323-39-3 / 29013-28-3

The in vitro genetic toxicity of Octadecanoic acid, monoester with 1,2-propanediol / Palmitic acid, monoester with 1,2-propanediol (CAS1323-39-3 / 29013-28-3)was assessed in a bacterial reverse mutation study (Ames test), performed under GLP conditions according to OECD guideline 471 (key, 2015). S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were exposed to the test substance at concentrations up to 5000 µg/plate. The vehicle and positive controls were valid in the presence and absence of metabolic activation. The test substance did not induce reversions in the S. typhimurium strains, with or without metabolic activation.

CAS1323-39-3

The in vitro genetic toxicity of Stearic acid, monoester with propane-1,2-diol (CAS 1323-39-3) was assessed in a bacterial reverse mutation study (Ames test) using S. typhimurium strains TA 1535, TA 1537, and TA 1538 at concentrations up to 5.0% (w/v) in agar (supporting, 1975). No cytotoxicity was seen. Vehicle, negative and positive controls were valid. In the presence and absence of metabolic activation the test substance did not induce reversions in the S. typhimurium strains.

CAS84988-75-0

The in vitro genetic toxicity of Fatty acids, C14-18 and C16-18-unsatd., esters with propylene glycol (CAS 84988-75-0) was assessed in a bacterial reverse mutation study (Ames test), performed under GLP conditions according to OECD guideline 471 (supporting, 1991). S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 1538 were exposed to the test substance at concentrations up to 5000 µg/plate. The vehicle, negative and positive controls were valid in the presence and absence of metabolic activation. The test substance did not induce reversions in the S. typhimurium strains, with or without metabolic activation.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 68171-38-0

An in vitro mammalian cell gene mutation assay was performed with Isooctadecanoic acid, monoester with propane-1,2-diol (CAS 68171-38-0) according to OECD guideline 490 (key, 2017). The main experiment consisted of three independent experiments (with 4 hours of exposure with and without metabolic activation and 24 hours of exposure without metabolic activation). All experiments were performed in mouse lymphoma L5178Y cells with test substance concentrations up to 80 μg/mL dissolved in DMSO. The dose levels were limited by test item induced toxicity. No precipitation of the test item was observed at the end of exposure. The positive and vehicle controls were valid and within the range of historical control data. No significant increase in mutation frequency was observed.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 68171-38-0

The cytogenetic potential of Isooctadecanoic acid, monoester with propane-1,2-diol (CAS 68171-38-0) was assessed in an in vitro mammalian chromosome aberration test in primary human lymphocytes, performed according to OECD guideline 473 (key, 2017). Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation. The pre-incubation period in cell medium was 48 h. Lymphocytes in whole blood cultures were exposed in duplicates to 4 hours with S9, 4 hours without S9, and 24 hours without S9. For each exposure condition tested concentrations were 0 (DMSO vehicle control), 20, 40, 60, 80, 100, 120, and 160 µg/mL. The maximum dose level tested in the main test was the lowest precipitating dose level; the dose range also included a dose level that induced greater than optimum toxicity with approximately 55±5% mitotic inhibition. Colcemid was added at 0.1 μg/mL at 2.5 hours before harvesting. 300 metaphases (150 per duplicate) per dose concentration were stained with Giemsa and counted for chromosome damage. Positive control substances were Mitomycin C for exposure without metabolic activation (S9 mix) and cyclophosphamide for exposure with S9 mix. All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The test item did not induce any statistically significant increases in the frequency of cells with aberrations.

 

Overall conclusion for genetic toxicity

The analogue read-across from source substances was applied from in vitro studies on gene mutation in bacterial cells. In vitro studies on cytogenicity and gene mutation in mammalian cells were available for the target substance. The results of the available studies were consistently negative. Based on the available data from analogue substances and the target substance, no mutagenic or clastogenic potential was found for Isooctadecanoic acid, monoester with propane-1,2-diol (CAS 68171-38-0).

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Isooctadecanoic acid, monoester with propane-1,2-diol (CAS 68171-38-0), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, including target substance data and the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.