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Genetic toxicity: in vitro

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Administrative data

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
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
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:

This data is being read across from the source study that tested Santicizer S278 (benzyl 3-(isobutyryloxy)-1-isopropyl-2,2-dimethylpropyl phthalate) based on analogue read across.

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
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

Test material

Constituent 1
Reference substance name:
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
EC Number:
701-008-3
Molecular formula:
C27H34O6
IUPAC Name:
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
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

Method

Species / strain
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
Controls
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).

Results and discussion

Test resultsopen allclose all
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)

Applicant's summary and conclusion

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.