Santicizer 278®

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Santicizer® 278 has shown no evidence of mutagenic activity in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 or TA1538, after exposure for at least 2 days at up to 10 mg/plate in the presence and absence of rat liver S9. Levels of 10 mg/plate were not toxic to any of the five strains in the presence or absence of S9, but levels of 3 mg/plate and higher exceeded the solubility of the test material (Environmental Health Laboratory, 1982).

 

The test material Santicizer® 278 did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested in a modern OECD 471 bacterial reverse mutation assay (Covance 2018). 

 

In a OECD 476 gene mutation assay in mammalian cells, Santicizer® 278 did not induce biologically relevant increases in mutant frequency at the hprt locus in mouse lymphoma L5178Y cells when tested up to precipitating concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9), under the experimental conditions (Covance 2018a).

 

Santicizer® 278 did not induce biologically relevant increases in micronucleus frequency in the frequency of micronuclei when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 24+24 hours in the absence of S-9 under the experimental conditions in a OECD 487 micronucleus assay (Covance 2019).

 

Therefore, following the completion of 3 further in vitro genotoxicity assays (OECD 471, OECD 476 and OECD 487) Santicizer® 278 has shown no evidence of mutagenic, clastogenic or aneugenic activity. 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 November 1981 to 25 January 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to OECD guidelines, with deviations

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Study did not utilise strains to detect cross-linking agents

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

as above

Principles of method if other than guideline:

Method: other: Ames et al. (1975). Mutat. Res. 31:347-364.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: S.typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

0.01, 0.04, 0.2, 1.0, 3.0, and 10 ul/plate (i.e. mg/plate)

Vehicle / solvent:

- Vehicle(s)/solvent(s): dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-N-oxide, 2-acetylaminofluorene, benzo(a)pyrene, sodium nitrite, 2-aminoanthracene, 9-aminoacridine,

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: at least 48 hours

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: Plates were examined visually for toxicity after at least 48 hours [no further details given on determination of cytotoxicity].

Evaluation criteria:

A positive response is indicated if three or more treatments on the initial test (and retest, if performed) are significantly greater than control (p<0.01) and if there is a significant positive dose-response (p<0.01) for the initial test (or retest, if performed).

Statistics:

Values of revertants/plate were transformed using log (base 10) for further analysis. Bartlett's test was performed to determine if a significant difference existed among treatment variables. Treatments were compared with controls using a one-sided t-test and within-levels pooled variance. Dose response was evaluated for all treatments which were not significantly lower (p<0.01) than controls, and used regression analysis for a log-log straight line. A t-test was used to evaluate significance of dose response.

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: levels of 3 mg/plate and higher exceeded the solubility limits of the test material

RANGE-FINDING/SCREENING STUDIES: In the toxicity screen, the test material was not toxic at up to 10 mg/plate in strain TA 100 (with and without S-9)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Conclusions:

Interpretation of results: negative

Santicizer(R) 278 showed no evidence of mutagenic activity in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or TA 1538 after exposure for at least 2 days at up to 10 mg/plate in the presence and absence of a rat liver metabolic activation system (S-9). Levels of 10 mg/plate were not toxic to any of the five strains in the presence or absence of S-9, but levels of 3 mg/plate and higher exceeded the solubility of the test material.

Executive summary:

Santicizer(R) 278 was examined for mutagenic activity in five strains of S. typhimurium using the plate incorporation test. The bacterial strains employed are capable of detecting both induced frameshift (TA 1537, TA 1538 and TA 98) and base-pair substitution (TA 1535 and TA 100) mutations. Tests were conducted in triplicate with the test material in DMSO at levels from 0.01 to 10 mg/plate, both with and without the addition of S-9. Revertant colonies per plate and toxicity were measured after at least 48 hours, and compared to the controls. Due to possible significant increases in revertants/plate over controls, two retests (again in triplicate) were conducted with the test material at 0.5, 1 and 1.5 mg/plate in S.typhimurium TA 100 (with S-9) and in TA 1537 (without S-9).

None of the test strain results had three treatments with revertants/plate significantly greater than controls and a significant dose response. No toxicity was seen in any of the five strains at up to 10 mg/plate in the presence or absence of S-9, although levels of 3 mg/plate and higher exceeded the solubility of the test material. The study authors concluded that the “plate incorporation test results indicated no significant mutagenic activity for this test material".

 

This study does not entirely conform with current OECD guidelines which recommend using S. typhimurium strain TA 102, or Escherichia coli WP2 uvrA to detect cross-linking agents. In additon, the study report does not describe how "toxicity" was determined, although it was probably based on a thinning of the background lawn of non-revertant cells.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March 2018 - 28 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test Material: Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate
CAS Number: CAS# 16883-83-3
Trade Name: Santicizer® 278
Batch number: 2984
Appereance: clear oily liquid
Received on: 02 March 2018
Stotage: 15-25°C protected from light
Purity: 98.51%
Expiry date: 31 October 2020 if unopened or 6 months from the date of opening

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

Mutation Experiment 1:
Treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test material at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, no evidence of toxicity was observed in the absence or presence of S-9.
An accurate assessment of toxicity was not possible at concentrations of 1600 µg/plate and above due to an oily layer of precipitation obscuring the background bacterial lawn.

Mutation Experiment 2:
Treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration was reduced to 1250 µg/plate based on precipitation observed in all strains in Mutation Experiment .
Narrowed concentration intervals were employed covering the range 3.0 - 1250 µg/plate in order to examine more closely those concentrations of the test material approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

TEST SYSTEM:
The test system was suitably labelled to clearly identify the study number, bacterial strain, test article concentration (where appropriate), positive and vehicle controls, in the absence or presence of S-9 mix.

METABOLIC ACTIVATION SYSTEM:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as
lyophilized S-9 mix (Mutazyme TM ), stored frozen at <-20°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert
ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities). Treatments were carried out both in the absence and presence of S-9 by addition of
either buffer solution or 10% S-9 mix respectively.

BACTERIA:
Five strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537 and TA102) were used in this study. For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate, to provide bacterial cultures in the range of approximately 10*8 to 10*9 cells/mL, based on cell count data from testing of each strain batch. Incubation was carried out with shaking in an anhydric incubator, set to turn on using a timer switch. All treatments were completed within 6 hours of the end of the incubation period.
The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline).

Evaluation criteria:

ACCEPTANCE CRITERIA:
The assay was considered valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges.
2. The positive control chemicals induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in
strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation.

EVALUATION CRITERIA
For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or
TA1537) the concurrent vehicle control values
2. Any observed response is reproducible under the same treatment conditions.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.

Statistics:

Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the laboratory’s historical control ranges. Data were considered acceptable if the vehicle control counts fell within the calculated
historical control ranges and the positive control plate counts were comparable with the historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 µg/plate).

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

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

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

The test material Santicizer® 278 did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study.

Executive summary:

The objective of this study was to evaluate the ability of the test material Benzyl 3 -isobutyryloxy-1 -isopropyl-2,2 -dimethylpropyl phthalate CAS# 16883 -83 -3 to induce reverse mutations in histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium in the presence and absence of rat liver metabolising system (S9).

The study was conducted according the OECD Guideline 471 and under GLP conditions.

During the Experiment 1, all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test material at 50, 160, 500, 1600 and 5000 µg/plate,

plus vehicle and positive controls. No evidence of toxicity was observed in the absence or presence of S-9, as would normally be manifest as a thinning of the background bacterial lawn or a marked reduction in revertant numbers. An accurate assessment of toxicity was not possible at concentrations of 1600 µg/plate and above due to an oily layer of precipitation.

During Experiment 2, the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration was reduced to 1250 µg/plate based on precipitation observed in all strains in Experiment 1.

Narrowed concentration intervals were employed covering the range 3.0 - 1250 µg/plate in order to examine more closely those concentrations of the test material approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, evidence of toxicity as a slight thinning of the background lawn was observed at 1250 µg/plate in strains TA100, TA1535, TA1537 and TA102 in the presence of S-9. An accurate assessment of toxicity was not possible at 1250 µg/plate in all strains in the absence of S-9 due to an oily layer of precipitation obscuring the background bacterial lawn. Precipitation was observed on the test plates at concentrations of 500 µg/plate and above (Experiment 1) or 625 µg/plate and above (Experiment 2), in the absence and presence of S-9.

Vehicles and positive controls were included for all strains in both experiments.

In conclusion, the test material Benzyl 3 -isobutyryloxy-1 -isopropyl-2,2 -dimethylpropyl phthalate CAS# 16883 -83 -3 did not induce mutation under the condition of this study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2018 - 31 May 2018

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)

Deviations:

no

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:

TEST MATERIAL NAME: Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate
CAS Number: 16883-83-3
Trading name: Santicizer® 278
Batch number: 2984
Appearance: clear oily liquid
Molecular weight: 454.4 g/mol
Received on: 02 March 2018
Storage Temperature: 15 25°C protected from light
Purity: 98.518%
Expiry date: 3 years from the date of manufacture if unopened (expiry of 21 December 2020) or 6 months from the date of opening (expiry of 02 October 2018)

The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor.

Species / strain / cell type:

lymphocytes: L5178Y tk+/- (3.7.2C) mouse lymphoma cells

Remarks:

NA

Details on mammalian cell type (if applicable):

CELLS: mouse lymphoma L5178Y cells

METABOLISING SYSTEM: in the absence and presence of a rat liver metabolising system (S-9)

ORIGIN CELL STOCK: Dr Donald Clive, Burroughs Wellcome Co.
CELL STORAGE CONDITIONS:Cells supplied to Covance were stored as frozen stocks in liquid nitrogen.

BACTERIAL CHECK: Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free.

Test concentrations with justification for top dose:

The concentrations used in the main test was based on the results on the Range Finder experiment.

The Range Finder concentrations were tested in the absence and presence of S-9 ranging from 9.375 to 300 µg/mL (limited by solubility in culture medium). Both immediately upon addition of the test article to the cultures and following the 3 hour treatment incubation period, precipitate was observed at the highest two concentrations tested in the absence and presence of S-9 (150 and 300 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and the higher concentration discarded. The highest concentration analysed (150 µg/mL), gave 106% and 97% RS, in the absence and presence of S-9, respectively .

No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations analysed (150 µg/mL) as compared to the concurrent vehicle controls.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

TEST SYSTEM:
The test system was suitably labelled to clearly identify the study number, test article concentration, positive and vehicle control, presence and absence of S 9.

CYTOTOXICITY RANGE FINDER:
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment volume was 20 mL.
Following 3 hour treatment, cells were centrifuged (200 g), washed with tissue culture medium and resuspended in 20 mL RPMI 10.
Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air for 9 days. Wells containing viable clones were identified by eye using background illumination and counted.

MUTATION ASSAY:
At least 107 cells in a volume of 17.8 mL of culture medium (RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours’ incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and finally resuspended in 20 mL RPMI 10 medium. Cell densities were determined using a Coulter counter and the concentrations adjusted to 2 x 105 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies (Brusick, 1986; Scott et al., 1991). Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.

PLATING FOR SURVIVAL:
Following adjustment of the cultures to 2 x 105 cells/mL after treatment, samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.

EXPRESSION PERIOD:
Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask.

PLATING FOR VIABILITY:
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (10 days). Wells containing viable clones were identified by eye using background illumination and counted.

PLATING FOR 6TG RESISTENCE:
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 105 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 µg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (14 days) and wells containing clones were identified as above and counted.

Evaluation criteria:

1. The MF in the vehicle control cultures was considered acceptable for addition to the laboratory historical negative control database
2. The MF in the concurrent positive controls induced responses that were comparable with those generated in the historical positive control database and gave a clear, unequivocal increase in MF over the concurrent negative control
3. The test was performed with and without metabolic activation
4. Adequate numbers of cells and concentrations were analysable.

Statistics:

ANALYSIS OF RESULTS:
All calculations were performed by computer using validated software.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Remarks on result:

other: The test material did not induce biologically relevant increases in mutant frequency at the hprt locus in mouse lymphoma L5178Y cells

Conclusions:

The test material Santicizer® 278 did not induce biologically relevant increases in mutant frequency at the hprt locus in mouse lymphoma L5178Y cells when tested up to precipitating concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9), under the experimental conditions described.

Executive summary:

The aim of the study was to investigate the ability of the test substance Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate CAS# 16883-83-3(also known as Santicizer®278) to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study was performed according to the OECD 476 Guideline and under GLP conditions.

A Range-Finder Experiment was performed followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254‑induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for each experiment. In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 9.375 to 300 µg/mL (limited by solubility in culture medium). The highest concentration analysed (150 µg/mL) was limited by the appearance of post treatment precipitate and gave 106% and 97% Relative Survival (RS), in the absence and presence of S-9, respectively. In the Mutation Experiment, nine concentrations, ranging from 5 to 300 µg/mL, were tested in the absence and presence of S-9. Seven days after treatment the highest concentration analysed for viability and 6TG resistance was 100 µg/mL in the absence and presence of S-9 (limited by the appearance of post treatment precipitate), which gave 85% and 83% RS, respectively. Vehicle and positive control treatments results were acceptable, therefore the study was accepted as valid.

Following 3 hour treatment in the absence and presence of S-9 no statistically significant increases in MF, compared to the vehicle control, were observed at any concentration analysed. All concentrations tested in the absence of S-9 were within the upper limit generated by the last twenty experiments performed in this laboratory (0.95 to 6.55 mutants per 10⁶viable cells). In the presence of S-9 concentrations of 10, 20 and 80 µg/mL were outside the range generated by the last twenty experiments (1.57 to 6.27 mutants per 10⁶viable cells). However, there were no statistically significant increases in MF at any concentration tested, the increases were not concentration related and were only marginally above the concurrent vehicle control (6.21 mutants per 106viable cells). These observations were therefore considered not biologically relevant.

In conclusion, the test material Santicizer®278 did not induce biologically relevant increases in mutant frequency at the hprt locus in mouse lymphoma L5178Y cells when tested up to precipitating concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9), under the experimental conditions described.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-04-2018 to 21-11-2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

TEST MATERIAL NAME: Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate
CAS Number: 16883-83-3
Trading name: Santicizer® 278
Batch number: 2984
Appearance: clear oily liquid
Molecular weight: 454.4 g/mol
Received on: 02 March 2018
Storage Temperature: 15 25°C protected from light
Purity: 98.518%
Expiry date: 3 years from the date of manufacture if unopened (expiry of 21 December 2020) or 6 months from the date of opening (expiry of 02 October 2018)

The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor.

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
Blood from two healthy, non-smoking Male volunteers from a panel of donors at Covance was used for each experiment.
- Suitability of cells:
The use of human peripheral blood lymphocytes is recommended because the cells are only used in short-term culture and maintain a stable karyotype (Evans & O’Riordan, 1975).
- Cell cycle length, doubling time or proliferation index:
Cell cycle length: 13 ± 2 hour
- Sex, age and number of blood donors if applicable:
Blood from two healthy, non-smoking Male volunteers (Range-Finder: 32, 32 years; Micronucleus Experiment 1: 32, 34 Years; Micronucleus Experiment 1: 28, 23 Years)
- Whether whole blood or separated lymphocytes were used if applicable:
human lymphocyte cultures prepared from the pooled blood of two male donors

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S9) prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Treatments covering a broad range of concentrations (Exp 1 - 3+21 (-S9) 2.500 to 50.00 µg/mL; Exp 1 - 3+21 (+S9) 10.00 to 180.0 µg/mL; Exp 1 - 24+24 (-S9) 2.500 to 50.00 µg/mL; Exp 2 - 3+21 (-S9) 10.00 to 300.0 µg/mL), separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations tested in the Micronucleus Experiment (limited by toxicity), were determined following a preliminary cytotoxicity Range-Finder Experiment (1.814 to 500.0 µg/mL).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
approximately 48 hours
- Exposure duration:
3 (±S9) or 24 hours (-S9)
- Fixation time (start of exposure up to fixation or harvest of cells):
-S9 (3 hour treatment): 72 hours; -S9 (24 hour treatment): 96 hours; + S9 (3 hour treatment): 72 hours

SPINDLE INHIBITOR (cytogenetic assays):
Cytochalasin B (Cyto-B (formulated in DMSO) added to post wash-off culture medium to give a final concentration of 6 µg/mL per culture.

STAIN (for cytogenetic assays):
Acridine Orange in phosphate buffered saline (PBS), pH 6.8

NUMBER OF REPLICATIONS:
2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, two to three minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried and stored protected from light at room temperature prior to staining. Slides were stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes and washed with PBS (with agitation) for a few seconds. The quality of the staining was checked. Slides were air-dried and stored protected from light at room temperature. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.

NUMBER OF CELLS EVALUATED:
A minimum ofone thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. For the 24 hour treatment in the absence of S9, an additional 1000 binucleate cells from each culture (therefore 4000 per concentration) were analysed from the test article concentrations selected for analysis.


CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
A micronucleus was only recorded if it met the following criteria:

1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
Relative index (RI)
- Any supplementary information relevant to cytotoxicity:
Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined. RI, which indicates the relative number of nuclei compared to vehicle controls was determined using the formula as follows:

RI = (number binucleate cells + 2 (number multinucleate cells) / total number of cells in treated cultures)

Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows:

Relative RI (%) = (RI of treated cultures / RI of vehicle controls) x 100

Cytotoxicity (%) is expressed as (100 – Relative RI)

A selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.

Rationale for test conditions:

Please see 'Any other information on materials and methods incl. tables' for information on Rationale for Test Conditions.

Evaluation criteria:

or valid data, the test article was considered to induce clastogenic and/or aneugenic events if:

1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed

2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed

3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).

The test article was considered positive in this assay if all of the above criteria were met.

The test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations (Thybaud et al., 2007).

Statistics:

After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.

The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989).

The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p ≤0.05 were accepted as significant.

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test material Santicizer® 278 did not induce biologically relevant increases in micronucleus frequency in the frequency of micronuclei.

Conclusions:

The test material Santicizer® 278 did not induce biologically relevant increases in micronucleus frequency in the frequency of micronuclei when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 24+24 hours in the absence of S-9 under the experimental conditions described.

Executive summary:

Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate a Reaction Mass of Benzyl (1R,1S) 2,2,4-trimethyl-1-[(2-methylpropanoyl)oxy]pentan-3-yl benzene-1,2- dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2-methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate (also known as Santicizer® 278) was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations tested in the Micronucleus Experiment (limited by toxicity), were determined following a preliminary cytotoxicity RangeFinder Experiment. Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of Santicizer® 278 on the replication index (RI). Micronuclei were analysed at three or four concentrations (Exp 1 - 3+21 (-S9) 25.00 to 50.00 µg/mL; Exp 1 - 3+21 (+S9) 80.00 to 160.00 µg/mL; Exp 1 - 24+24 (-S9) 25.00 to 50.00 µg/mL; Exp 2 - 3+21 (-S9) 10.00 to 40.00 µg/mL)).

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The mean proportion of micronucleated binucleate (MNBN) cells in these cultures fell within the 95th percentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei. All acceptance criteria were considered met and the study was therefore accepted as valid.

Following 3+21 hour treatment with Santicizer® 278 in the absence of S9, frequencies of MNBN cells were similar to and not significantly higher (at the p≤0.05 level) than those observed in concurrent vehicle controls at all but the lowest concentration analysed (25 µg/mL). The MNBN cell frequency of all Santicizer® 278 treated cultures fell within the normal ranges with the exception of single replicates at the lowest and highest concentrations analysed (25 and 50 µg/mL). The sporadic increases were not concentration related and were therefore considered of no biological relevance. However, the maximum concentration analysed did not result in a 50-60% reduction in replication index (RI, which indicates the relative number of cell cycles per cell during the period of exposure) as required by the OECD guideline and therefore a confirmatory experiment was performed (Experiment 2).

Following 3+21 hour treatment in the absence of S9 in Experiment 2 four vehicle control replicates and an additional 2000 cells per concentration were analysed to aid data interpretation. Frequencies of MNBN cells were similar to and not significantly higher (at the p≤0.05 level) than those observed in concurrent vehicle controls at all concentrations analysed. The MNBN cell frequency exceeded the normal range in both replicates at the lowest two concentrations analysed (10 and 25 µg/mL) and in a single replicate at the highest concentration analysed (40 µg/mL). In addition, two of the four vehicle control replicates also exceeded the normal range. However, the mean MNBN cell frequency for the vehicle control was within range and the data were therefore considered acceptable. The increases observed in the test article treated cultures were not statistically significant and there was no statistical evidence of a linear trend indicating that the increases were not concentration related. According to data interpretation strategies proposed by Thybaud et al (Strategy for genotoxicity testing: Hazard identification and risk assessment in relation to in vitro testing. Mutation Research 627, 41-58, 2007), non-reproducible or marginal responses may be considered of very low or no toxicological concern and no further testing should be needed. Based on the lack of statistical significance, the magnitude of the increases observed and the poor reproducibility between experiments, the increases were considered of no biological relevance.

Following 3+21 hour treatment with Santicizer® 278 in the presence of S9 frequencies of MNBN cells were similar to and not significantly higher (at the p≤0.05 level) than those observed in concurrent vehicle controls at all concentrations analysed. The MNBN cell frequency of all treated cultures fell within normal ranges with the exception of a single replicate at the highest concentration analysed (160 µg/mL). There was no significant linear trend, indicating that the response was not concentration related and the increase was not well reproduced between the replicate cultures and was therefore considered of no biological relevance.

Following 24+24 hour treatment with Santicizer® 278 in the absence of S9 frequencies of MNBN cells were similar to and not significantly higher (at the p≤0.05 level) than those observed in concurrent vehicle controls at all concentrations analysed. Sporadic increases in MNBN cell frequency were observed in single replicates at the lowest two concentrations analysed (25 and 35 µg/mL) and both replicates were outside normal range at the highest concentration analysed (50 µg/mL). There was no concentration relationship (evidenced by a non-significant linear trend) and no statistical significance in the test article treated cultures. The criteria for a positive result were not fulfilled and the observations were therefore considered of no biological relevance.

It should be noted that the highest concentrations analysed following 3+21 hour treatment in the presence of S9 and 24+24 hour treatment in the absence of S9 gave a 67% reduction in RI (in excess of the recommended toxicity limit). However, as no biologically relevant increases in MNBN frequency were observed for either treatment condition, the data are considered acceptable.

It is concluded that Santicizer® 278 did not induce biologically relevant increases in the frequency of micronuclei in cultured human peripheral blood lymphocytes when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S9) and for 24+24 hours in the absence of S9 under the experimental conditions described. The maximum concentration analysed under all treatment conditions was limited by toxicity.

Additional information

A number of structurally related phthalates have shown no evidence of mutagenicity in the Ames test. Thus, di-isononyl phthalate (DINP) and butyl benzyl phthalate (BBP) were both inactive in the same fiveS. typhimuriumstrains when tested at up to 5-12 mg/plate in the presence or absence of a rat liver metabolic activation system (S9) (Environmental Health Laboratory, 1976; Litton Bionetics Inc., 1976; McKee et al. 2000; NTP, 1997d). Negative Ames test results have also been obtained with a number of other high molecular-weight phthalate esters (HMWPEs, with a carbon backbone of C7 or higher) (OECD, 2004. Draft SIDS Initial Assessment Report on High Molecular Weight Phthalate Esters (HMWPE) category.).

 

Other in vitro genotoxicity assays on various phthalates also provide reassuring findings. For example, DINP was inactive in a chromosome aberration test in Chinese hamster ovary (CHO) cells, when tested at up to 160 µl/ml in the presence or absence of rat liver S9 (McKee et al. 2000). Other HMWPEs have been found to be inactive in tests for chromosome aberrations or polyploidy in Chinese hamster lung cells and for mutagenicity in mouse lymphoma tests (OECD, 2004). BBP has shown no convincing evidence of genotoxic activity in chromosome aberration or sister-chromatid exchange (SCE) tests in Chinese hamster ovary cells (NTP, 1997e) or in the mouse lymphoma assay (Litton Bionetics, 1977; NTP, 1997c).

 

DINP and di-isodecyl phthalate (DIDP) did not induce chromosome damage (increased frequency of micronuclei) in the bone marrow erythrocytes of male mice (5/dose) receiving up to 2 and 5 g/kg bw/day, respectively, by stomach tube on 2 consecutive days (McKee et al. 2000; OECD, 2004).

 

BBP has shown only weak genotoxic potential in vivo, in chromosome aberration and SCE assays, following a single intraperitoneal injection of up to 5 g/kg bw in mice (NTP, 1997f). Two other in vivo studies of lower reliability provide no evidence of genotoxic potential. Thus, in a study available in abstract form only (reliability 4), BBP did not induce dominant lethal mutations in the foetuses when two strains of male mice were given three subcutaneous injections of up to 4.6 g/kg bw and mated with untreated females of the same strain. A slight, dose-related reduction in total implants was observed after the first mating period, indicating that the compound was tested at a high enough dose to produce signs of toxicity (a slight reduction in fertility) (Bishop et al., 1987). Another study assigned a reliability code of 3 (not reliable) due to the low test dose used (0.18 mg/kg bw/day) found no evidence of micronucleus induction in the bone marrow of female rats that were treated via the drinking water throughout pregnancy and lactation (Ashby et al. 1997).