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REACH

Reaction mass of bis(2-ethylhexyl) terephthalate, butyl 2-ethylhexyl terephthalate, and dibutyl terephthalate

EC number: 946-149-3 | CAS number: 1571954-81-8

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance, GL500, did not show any evidence to induce chromosomal aberrations in Chinese Hamster Lung (CHL/IU) cell under the conditions of the Chromosone aberration study.

GL500 was considered to be non-mutagenic under the conditions of the Ames study.

The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: in vitro gene mutation study in bacteria
Remarks:: Type of genotoxicity: chromosome aberration
Type of information:: experimental study
Adequacy of study:: key study
Study period:: This study was conducted between 03 July 2013 and 15 July 2013
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:: 1997
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:: EC No. 440/2008 of 30 May 2008
Deviations:: no
Qualifier:: according to guideline
Guideline:: other: 40 CFR 799.9510 TSCA bacterial reverse mutation test.
Deviations:: no
Qualifier:: according to guideline
Guideline:: other: USA, EPA (TSCA) OCSPP harmonized guidelines.
Deviations:: no
Qualifier:: according to guideline
Guideline:: other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:: no
GLP compliance:: yes
Type of assay:: bacterial reverse mutation assay
Specific details on test material used for the study:: Identification: Batch:vGL500 20130510
Purity: 99.81%
Expiry Date: 10 May 2018
Storage Conditions: Room temperature in the dark
No correction was made for purity was made
Target gene:: All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TAl 00, the R-factor plasmid pK.MlOl enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
Species / strain / cell type:: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:: not applicable
Metabolic activation:: with and without
Metabolic activation system:: The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/ -Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4.
Test concentrations with justification for top dose:: Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 50 to 5000 µg/plate.
Vehicle / solvent:: The vehicle control used was acetone
Negative solvent / vehicle controls:: yes
Remarks:: Identity: Dimethyl sulphoxide Supplier: Fisher Scientific Batch number (purity): 1684307 (>99%) Expiry: 02/2022 1670196 (>99%) Expiry: 03/2022 (Experiment 2 only)
Positive controls:: yes
Positive control substance:: other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:: Microsomal Enzyme Fraction
Lot No. PB/13NF S9 14 April 2013 was used in this study. The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/ -Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (lg liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

S9-Mix and Agar
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Media
Top agar was prepared using 0.6% Bacto agar (lot number 2299373 09/17) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 34519 07/13, 34498 07/13 and 34520 07/13).

Test System
Tester strains
The five strains of bacteria used, and their mutations, are as follows:

Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor

Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).

The bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Stateboume liquid nitrogen freezer, model SXR 34.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot numbers 1218742 07/17 and 1253889 10/17) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Test and Control Item Preparation
Test Item
The test item was immiscible in dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle. Following solubility information provided by the Sponsor, sterile distilled water was not evaluated as a potential vehicle in this test system.
The test item was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. Acetone is toxic to the bacterial cells at
0.1 mL (100 µL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 µL) aliquots. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.

Control Items

Vehicle and positive controls were used in parallel with the test item. The vehicle control used was acetone.
The positive control items used in the series of plates without S9-mix were as indicated in "Any other information... (below):

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, vehicle or appropriate positive control was added to 2 mL of trace amino-acid supplemented media (at approximately 45 °C) containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Each concentration of the test item, appropriate positive control, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the trace amino-acid supplemented media instead of phosphate buffer.

All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 50 to 5000 µg/plate.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the test item formulation or solvent or 0.1 mL of appropriate positive control were incubated at 37 °C± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 °C ± 3 °C for 20 minutes (with shaking) and addition of amino-acid supplemented media. All testing for this experiment was performed in triplicate.

All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:

All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).

All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the General Study Plan, Section 2.2.2 (negative controls).

All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.

Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.

There should be a minimum of four non-toxic test item dose levels.

There should be no evidence of excessive contamination.
Evaluation criteria:: Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal
Statistics:: Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control
: Key result
Species / strain:: other: All bacterial strains tested
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: no cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Remarks:: These data are for concurrent untreated control plates performed on the same day as the Mutation Test
Positive controls validity:: valid
Additional information on results:: Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test and consequently the same maximum dose level was used in the second mutation test. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test. A test item precipitate (globular in appearance) was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:: other: GL500 was considered to be non-mutagenic
Conclusions:: GL500 was considered to be non-mutagenic under the conditions of this test.
Executive summary:: The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method Bl3/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, 40 CFR 799.9510 TSCA bacterial reverse mutation test and the USA, EPA (TSCA) OCSPP harmonized guidelines.



Methods

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TAlOO andEscherichia colistrainWP2uvrAwere treated with the test item using both the Ames plate incorporation and pre incubation methods at up to eight dose levels, in triplicate, both with and without the addition of  a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 50 to 5000µg/plate.



Results

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix werevalidated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test and consequently the same maximum dose level was used in the second mutation test. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test. A test item precipitate (globular in appearance) was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertantcolonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubationmethod).

Conclusion

GL500 was considered to be non-rnutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 27 June 2013 and 02 October 2013

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:

21 July 1997

Deviations:

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Test Substance

Identity GL500
Lot No. 20130510
Appearance Transparent liquid
Ingredients Dibutyl terephthalate, Butyloctyl terephthalate, Dioctyl terephthalate
Molecular weight Mean : 362.5
Purity 99.81%
Affinity Hydrophobic, lipophilic
Date of manufacture May 10, 2013
Expiration date May 10, 2018
Storage condition Room temperature (17.2 – 21.7°C)
Handling instruction Not specified
Supplier:
Name LG Chem, Ltd.
Address LG Twin Towers 20, Yeouido-dong, Yeongdeungpo-gu, Seoul, 150-721, Korea
Date of receipt June 5, 2013

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The Chinese Hamster Lung cell line (Lot No.: 3375917) was purchased from American Type Culture Collection (ATCC, U.S.A.) on Nov. 24, 2011. Cells were seeded in a 75 cm2 cell culture flask (Nunc, Denmark) containing Eagle’s Minimum Essential Medium (Eagle’s MEM, Lonza Walkersville Inc., U.S.A.) supplemented
with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, U.S.A.) and incubated at 37°C with 5% CO2 (MCO-20AIC, SANYO, Japan). Cells were evaluated for contamination of mycoplasma by Hoechst Stain Kit (MPBIOMEDICALS, Japan).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 and Cofactor C were purchased from ORIENTAL YEAST Co., LTD in Japan and stored at -80 to -60°C and used within the expiration date

Test concentrations with justification for top dose:

The final concentrations to which cells were exposed initially are given below. Those cultures that were analysed for chromosome aberrations areindicated *.
Preliminary toxicity test: 12.09, 20.16, 33.59, 55.99, 93.31, 155.52, 259.2, 432, 720 and 1200 g/mL
Main tests: -S9 mix (3 hours) 20*, 25, 30*, 35, 40*, 45 and 50 µg/mL
+S9 mix (3 hours) 90, 100, 110, 120, 130, 140 and 150 µg/mL
-S9 mix (21 hours) 5*, 10, 15, 20*, 22.5*, 25, 27.5 and 30 µg/mL
Additional Main test: +S9 mix (3 hours) 100, 110, 120, 130, 140*, 150, 160*, 170, 180, 190* and 200 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

Positive controls:

yes

Positive control substance:

mitomycin C

Untreated negative controls:

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

PREPARATION OF CULTURES:
Cell Line: Chinese Hamster Lung (CHL/IU) cells

Justification for selection
The Chinese Hamster Lung (CHL/IU) cell line is routinely used in in vitro chromosome aberration studies and is required in the guidelines.

Supplier and storage
The Chinese Hamster Lung cell line (Lot No.: 3375917) was purchased from American Type Culture Collection (ATCC, U.S.A.) on Nov. 24, 2011. Cells were seeded in a 75 cm2 cell culture flask (Nunc, Denmark) containing Eagle’s Minimum Essential Medium (Eagle’s MEM, Lonza Walkersville Inc., U.S.A.) supplemented
with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, U.S.A.) and incubated at 37°C with 5% CO2 (MCO-20AIC, SANYO, Japan). Cells were evaluated for contamination of mycoplasma by Hoechst Stain Kit (MPBIOMEDICALS, Japan).
One mL of 0.25% Trypsin-EDTA was added to the cell culture flasks to detach cells from the bottom. Suspended cells were harvested, placed in a 50 mL plastic centrifuge tube and centrifuged at 1,000 rpm for 5 minutes. The supernatant was decanted and the pellets were resuspended with an appropriate amount of fetal bovine serum (FBS) to yield a concentration of 1×106 cells/mL. DMSO (Merck,
Germany) was added to a final concentration of 10%. Approximately 1 mL of cell suspensions was transferred to sterile cryogenic vials, stored at -80 to -60°C for 24 hours and stored in a liquid nitrogen tank until use.

Sub-culture
Frozen cells were thawed in a water bath 37°C and transferred to 50 mL plastic centrifuge tubes containing EMEM supplemented with 10% FBS and centrifuged at 1,000 rpm for 5 minutes. The supernatant was decanted and the pellet was resuspended with EMEM supplemented with 10% FBS. Suspended cells were transferred to a 75 cm² culture flask and incubated in a 5% CO2 incubator at 37°C.
Cell morphology was evaluated following 70–80% proliferation on the bottom of the flask. 0.25% Trypsin-EDTA was added to detach cells. The suspended cells were harvested, transferred to a 50 mL plastic centrifuge tube and centrifuged at 1,000 rpm for 5 minutes. The supernatant was decanted and the pellet was resuspended with EMEM supplemented with 10% FBS. Suspended cells were transferred to a 75 cm² culture flask and incubated in a 5% CO2 incubator at 37°C.

Pre-incubation
Cells within 24 passages were used in this study. Exponentially growing stock cultures were treated with trypsin-EDTA solution to separate cells from the bottom of culture flasks. The harvested cells were placed in a 50 mL plastic centrifuge tube and centrifuged at 1,000 rpm for 5 minutes. The supernatant was decanted and the pellets was resuspended in an appropriate volume of EMEM at 5×104 cells/mL and
placed in a 96 well plate (200 μL/well; Nunc, Denmark) for the growth inhibition study and a 60 mm dish (5 mL/plate, BD, U.S.A.) for the main study. Cells were incubated in a 5% CO2 incubator at 37°C for one day.

S9 metabolizing system
Preparation of S9 fraction
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from ORIENTAL YEAST Co., LTD in Japan and stored at -80 to -60°C and used within the expiration date..

Preparation of S9 mix
Preparation of S9 mix was conducted immediately prior to use. The frozen S9 (Lot No.: 13012511) and Cofactor C (Lot No.: C13012311) were mixed at a ratio of 2 to 4.7.

Growth Inhibition Study
It was conducted to determine the high dose for the main study under the non-GLP.
Dose levels
The high dose of the test substance was 3,620 μg/mL (approximately 10 mM), the limit dose recommended in the guideline. The high dose was sequentially diluted to produce lower dose levels (1,810, 905, 453, 226, 113, 56.6, 28.3 and 14.1 μg/mL). The negative control group was used.
Method
After pre-incubation, all plates were divided such as short time treatment with and without metabolic activation and the continuous treatment without metabolic activation. Four wells per dose were evaluated. The identification numbers were recorded on all plates.
Each well for the short time treatment with and without metabolic activation was washed with Dulbecco’s phosphate-buffered saline (D-PBS) after 6 hours treatment. Then a fresh medium was added and cultured for an additional 18 hours. In the continuous treatment without metabolic activation, cells were treated for 24 hours.
In both short time treatment with and without metabolic activation and the continuous treatment without metabolic activation, cells were incubated in a 5% CO2 incubator at 37°C.
The deposition of the test substance was evident after addition of test substance and the end of treatment
Measurement of absorbance
Following 24 hours (culture completion time), 50 μL of 3-[4, 5-dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL D-PBS) was added and cultured for 4 hours. After cultivation, the wells were dried and DMSO (150 μL/well) was added. The spectrophotometric absorbance of the sample was measured using an ELISA reader (ELx808IU, BioTek, U.S.A.) at 540 nm.
The results were expressed in graphic form and the absorbance and calculated median growth inhibitory concentration (IC50) were calculated and reported.

Justification for selection main study dose level

Results of the growth inhibition study
Cytotoxicity was not evident in the short time treatment with and without metabolic activation and the continuous treatment without metabolic activation.
The deposition of the test substance was evident at more than 453 μg/mL in the short time treatment with metabolic activation. In the short time treatment without metabolic activation and the continuous treatment without metabolic activation, it was not evident at all dose levels.

Dose levels of the main study
Based on the results of the growth inhibition study, the high dose of the main study was selected at 453 μg/mL, which was the limit of solubility in the final culture medium at the end of the treatment period, in the short time treatment with metabolic activation. In the short time treatment without metabolic activation and the continuous treatment without metabolic activation, it was selected at 3,620 μg/mL because cytotoxicity was not evident at all dose levels. These high dose level was sequentially diluted by a geometric ratio of 2 to produce 2 additional lower dose levels.

Main Study
1st study
After pre-incubation, all plates were divided into two groups: short time treatment with and without metabolic activation. The identification numbers were recorded on the top and bottom of the culture plates. Two plates were prepared for each group.
Each plate was washed with D-PBS after 6 hours treatment. Then a fresh medium was added and cultured for an additional 18 hours.
In both short time treatment with and without metabolic activation, cells were incubated in a 5% CO2 incubator at 37°C.
The deposition of the test substance was evident after addition of test substance and the end of treatment.

2nd study
After pre-incubation, all plates were divided into two groups: short time treatment with metabolic activation and continuous treatment without metabolic activation. The identification numbers were recorded on the top and bottom of the culture plates. Two plates were prepared for each group.

Each plate for the short time treatment with metabolic activation was washed with D-PBS after 6 hours treatment. Then a fresh medium was added and cultured for an additional 18 hours. In the continuous treatment without metabolic activation, cells were treated for 24 hours.
In both short time treatment with metabolic activation and the continuous treatment without metabolic activation, cells were incubated in a 5% CO2 incubator at 37°C.
The deposition of the test substance was evident after addition of test substance and the end of treatment.

Slide preparation
Two hours prior to culture completion 0.2 μg/mL of colcemid (Invitrogen, U.S.A.) was added to arrest cells in metaphase. Following culture completion, cells were treated with 0.25% trypsin, centrifuged (FLEAT 5, Hanil Science Industrial Co.,Ltd.) at 1,000 rpm for 5 minutes and incubated in 0.075 mol/L KCl hypotonic solution at 37°C for 20 minutes. After incubation in the hypotonic solution, cells were fixed with 1 mL of ice- cold fixative (methanol : acetic acid, 3 : 1) and centrifuged at 1,000 rpm for 5 minutes. The supernatant was decanted and cells were fixed with 5 mL of ice-cold fixative and centrifuged at 2,000 rpm for 5 minutes. This procedure was repeated once. One or two drops of the suspension were placed on clean dry slides. The slides were identified with random numbers. The slides were air-dried and stained with 3% Giemsa solution (Giemsa, Sigma-Aldrich, U.S.A., in 0.01 mol/L Sörenson phosphate buffer, pH 6.8) for 20 minutes.

Observations

Observation of slides was conducted from the 1st study to the 2nd study in order.
Chromosomal evaluations were conducted as follows; the high dose level, at which 200 metaphases can be observed, and lower dose levels was selected at least 3 dose levels for chromosome evaluations in the short time treatment with and without metabolic activation and continuous treatment without metabolic activation.
In each coded slide, well-spread 100 metaphase cells per plate (200 metaphases/dose) was evaluated using a microscope (BX51, Olympus, Japan) at a magnification of 400 fold.
Structural chromosomal aberrations were classified into chromatid break (ctb), chromatid exchange (cte), chromosome break (csb), chromosome exchange (cse), chromatid or chromosome gap (gap) and other (o). When several gaps or breaks were evident in metaphase, these were recorded as a fragment (frg) in other (o). Gaps (g) were not recorded as structural aberrations and were not included in the calculation of the aberration rates. An achromatic lesion narrower than the width of one chromatid was recorded as a gap. The frequency of numerical aberrations (polyploid; pol, endoreduplication ; end) was recorded.
For structural and numerical aberrations, any cell with one or more aberrations will be counted as one aberrant cell.

Acceptance Criteria

Evaluation of the validity of the study was conducted based on the following criteria:

･ The frequency of cells with chromosome aberrations in the negative and positive control groups is below 5% and above 10%, respectively.
･ The dose levels which have more than 200 metaphases per dose are above three.
･ The cultures do not show any evidence of contamination.

Evaluation Criteria

The frequencies of chromosome aberration cells except gaps were determined in accordance with the criteria of Toshio Sofuni as follows:

Mean frequencies of chromosome aberration cells Evaluation
Below 5% Negative (-)
Above 5% – below 10% Equivocal positive (±)
Above 10% Positive (+)

The result of the main study was negative, D20 and TR values were not calculated and reported.
- D20 : The minimum dose (mg/mL) at which aberrations are found in 20% of metaphase
- TR value: comparison value of chromatid exchange (cte) per mg/mL

Evaluation criteria:

An assay was considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance was considered to cause a positive response if the following conditions were met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) were observed at one or more test concentration.
The increases exceed the vehicle control range of this laboratory, taken at the 95% confidence limit.
The increases were reproducible between replicate cultures.
The increases were not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response was considered to support the conclusion.
A negative response was claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
A further evaluation would be carried out if the above criteria for a positive or a negative response are not met.

Statistics:

Statistical analysis was performed using SAS Program (version 9.3, SAS Institute Inc., U.S.A.).
Since the result was negative, for the aberration cell data, Fisher’s exact test was used for comparison of the negative control group to the test substance groups or the positive control group (significance level: 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Deposition of the Test Substance

The deposition of the test substance was evident at 453 μg/mL in the short time treatment with metabolic activation. In the short time treatment without metabolic activation and the continuous treatment without metabolic activation, it was not evident at all dose levels.

Selection of Dose Levels for Observation of Chromosome Aberrations (Tables 1 and 2)
In the slide preparation, 200 metaphase cells were evident at the high dose levels of the short time treatment with and without metabolic activation and the continuous treatment without metabolic activation. Therefore, successive three dose levels containing the high dose were selected for observation of chromosome aberrations.

Treatment S9 mix Dose Levels for Observation of Chromosome Aberrations (μg/mL)
Short time - 3,620, 1,810, 905
+ 453, 226, 113
Continuous - 3,620, 1,810, 905

The Frequency of Cells with Chromosome Aberrations (Tables 1 and 2)

The frequency of cells with chromosome aberrations in the short time treatment with and without metabolic activation and in the continuous treatment was less than 5% at all dose levels. Also, no statistically significant differences were noted in the test substance groups when compared to the negative control group (Fisher’s exact test, p≥0.05).
In the positive control group, the frequency of cells with structural chromosome aberrations was statistically significantly increased when compared to that in the negative control group (Fisher’s exact test, p<0.05).

Acceptance of Study

The frequency of cells with chromosome aberrations in the negative and positive control groups was below 5% and above 10, respectively. In addition, the dose levels which had more than 200 metaphases per dose (200 metaphases/dose) were three dose levels. Also, there were no contaminations in cultures

Remarks on result:

other: No evidence toinduce chromosomal aberration in Chinese Hamster Lung cells

Table1 Summary of First Study

Test substance	Dose(μg/mL)	S9 mix	Trt-RecTime(hr)	No. of cellanalyzed	Number of cells with structural aberrations						gap (%)	Number of cells withnumerical aberrations
					ctb	csb	cte	cse	frg	total (%)		end	pol	total (%)
Acetone	0	-	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	1 (0.5)
				100	0	0	0	0	0			0	1
GL500	905	-	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	2 (1.0)
				100	0	0	0	0	0			0	2
	1,810	-	6-18	100	1	0	0	1	0	2 (1.0)	0 (0.0)	0	2	2 (1.0)
				100	0	0	0	0	0			0	0
	3,620	-	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)	0	1	2 (1.0)
				100	0	0	0	0	0			0	1
MMC	0.1	-	6-18	100	9	1	17	0	0	54* (27.0)	0 (0.0)	0	1	1 (0.5)
				100	19	0	16	0	0			0	0
Acetone	0	+	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)	0	1	1 (0.5)
				100	0	0	0	0	0			0	0
GL500	113	+	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)
				100	0	0	0	0	0			0	0
	226	+	6-18	100	1	0	0	0	0	1 (0.5)	0 (0.0)	0	0	0 (0.0)
				100	0	0	0	0	0			0	0
	453†	+	6-18	100	0	0	1	0	0	1 (0.5)	1 (0.5)	0	0	0 (0.0)
				100	0	0	0	0	0			0	0
B[a]P	20	+	6-18	100	6	0	17	0	0	36* (18.0)	0 (0.0)	0	2	2 (1.0)
				100	5	0	12	0	0			0	0

Table 2. Summary of Second Study

Test substance	Dose(μg/mL)	S9 mix	Trt-RecTime(hr)	No. of cellanalyzed	Number of cells with structural aberrations						gap (%)	Number of cells withnumerical aberrations
					ctb	csb	cte	cse	frg	total (%)		end	pol	total (%)
Acetone	0	+	6-18	100	0	0	0	0	0	0 (0.0)	1 (0.5)	0	0	0 (0.0)
				100	0	0	0	0	0			0	0
GL500	113	+	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)
				100	0	0	0	0	0			0	0
	226	+	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)
				100	0	0	0	0	0			0	0
	453†	+	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)
				100	0	0	0	0	0			0	0
B[a]P	20	+	6-18	100	8	1	12	0	0	32* (16.0)	0 (0.0)	0	1	1 (0.5)
				100	10	0	11	0	0			0	0
Acetone	0	-	24-0	100	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)
				100	0	0	0	0	0			0	0
GL500	905	-	24-0	100	0	0	0	0	0	0 (0.0)	1 (0.5)	0	0	0 (0.0)
				100	0	0	0	0	0			0	0
	1,810	-	24-0	100	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	1 (0.5)
				100	0	0	0	0	0			0	1
	3,620	-	24-0	100	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)
				100	0	0	0	0	0			0	0
MMC	0.1	-	24-0	100	19	0	25	0	0	81* (40.5)	0 (0.0)	0	0	0 (0.0)
				100	11	0	34	0	0			0	0

Aberration;gap:chromatidandchromosomegap,ctb:chromatidbreak,cte:chromatidexchange,csb:chromosomebreak,cse:chromosome exchange,frg: fragmentation, end:endoreduplication, pol:polyploidy

MMC:MitomycinC,B[a]P:Benzo[a]pyrene Trt-Rectime:Treatment-Recoverytimes

Significant difference from negative control by fisher's exact test : * p<0.05

† : Deposition

Conclusions:

The test substance, GL500, did not show any evidence to induce chromosomal aberrations in Chinese Hamster Lung (CHL/IU) cell under the conditions of this study.

Executive summary:

This study was designed to evaluate the potential of the test substance, GL500, to produce chromosomal aberrations in Chinese Hamster Lung (CHL/IU) cells.

In order to determine the high dose level of main study, the growth inhibition study was conducted. As a result, cytotoxicity was not evident in the short time treatment with and without metabolic activation and the continuous treatment without metabolic activation.

The deposition of the test substance was evident at more than 453 μg/mL in the short time treatment with metabolic activation. In the short time treatment without metabolic activation and the continuous treatment without metabolic activation, it was not evident at all dose levels.

Based on the results of the growth inhibition study, the high dose of the main study was selected at 453 μg/mL, which was the limit of solubility in the final culture medium at the end of the treatment period, in the short time treatment with metabolic activation. In the short time treatment without metabolic activation and the continuous treatment without metabolic activation, it was selected at 3,620μg/mL.

Therefore, the following dose levels were selected for the main study.

Treatment	S9 mix	Dose levels of the main study (μg/mL)
Short time	-	3,620, 1,810, 905
Short time	+	453, 226, 113
Continuous	-	3,620, 1,810, 905

Also, the positive and negative control groups were used.

As results of mainstudy,the frequency of cells with chromosome aberrations was less than 5% in the short time treatment with and without metabolic activation and the continuous treatment without metabolicactivation.

In the positive control group, the frequency of cells with structural chromosomal aberrations was statistically significantly increased compared to that in the negative control group.

Based on the results of this study, the test substance, GL500, did not exhibit the chromosome aberrations under the conditions of this study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: V79 HPRT

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 15 September 2016 and 16 December 2016

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)

Version / remarks:

28 July 2015

Deviations:

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

Test Substance

IIdentification: GL500
Physical State / Appearance: Clear colourless liquid
Alternative Names: LGflex GL500, GL500, GL520
Batch: GLFG160607
CAS No.: 1571954-81-8
Purity: 99.8%
Expiry Date: 07 June 2017
Storage Conditions: Room temperature, in the dark
Intended use / Application: Plasticizer

No correction for purity was made.

Target gene:

the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Lot Number 25/08/16 was used in this study, and was pre-prepared in house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 tested for its capability to activate known mutagens in the Ames test.

Test concentrations with justification for top dose:

The concentrations of test item used in the preliminary cytotoxicity test were 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL.

Vehicle / solvent:

The vehicle control used was as follows:
Identity: Acetone
Supplier: Fisher Scientific
Batch Number: 1664439 (Preliminary Toxicity Test) 1664439 (Main Experiment)

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Controls

Vehicle and positive controls were used in parallel with the test item. The vehicle control used was as follows:
Identity: Acetone
Supplier: Fisher Scientific
Batch Number: 1664439 (Preliminary Toxicity Test) 1664439 (Main Experiment)

The positive control items were as follows: Absence of S9-mix:
Identity: Ethyl methane sulphonate (EMS)
Supplier: Sigma Aldrich
Batch Number: BCBQ451V
Purity: Assume 100%
Expiry Date : 12 February 2017
Solvent: DMSO
Concentration: 500 and 750 µg/mL for 4-hour exposure

Presence of S9-mix:
Identity: Dimethyl benzanthracene (DMBA)
Supplier: Sigma Aldrich
Batch number: SLBM4575V
Purity: 95%
Expiry Date : 06 April 2017
Solvent: DMSO
Concentration: 1 and 2 µg/mL for 4-hour exposure

Test System and Supporting Information
Cell Line
The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).

The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.

Cell Culture
Laboratory stock cell cultures will be periodically checked for stability and absence of mycoplasma contamination. The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37”C with 5% CO2 in humidified air.

Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 µg/mL, 100 µM). Aminopterin (0.0178 µg/mL, 0.4 µM) and Thymidine (3.85 µg/mL, 16 µM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Microsomal Enzyme Fraction
Lot Number 25/08/16 was used in this study, and was pre-prepared in house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 tested for its capability to activate known mutagens in the Ames test.

The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G-6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.

Experimental Design and Study Conduct
Test Item Preparation
The test item was considered to be a UVCB* and therefore the maximum recommended dose concentration was 5000 µg/mL. However, due to the necessity of using acetone as the solvent, the maximum concentration that could be achieved was 2500 µg/mL. The purity of the test item was 99.8% and was therefore tested with no purity correction applied to the formulations. (* UVCB = Substances of Unknown or Variable composition, Complex reaction products or Biological materials.)

The test item was immiscible in culture medium at 50 mg/mL and immiscible in dimethyl sulfoxide at 500 µg/mL. The test item was miscible in acetone at 500 mg/mL which allowed a maximum achievable dose of 2500 µg/mL, when dosed at 0.5% of the culture volume.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the concentrations investigated (Scott et al., 1991). The pH and osmolality readings are presented in the following table:

Concentration µg/mL 0 9.77 19.53 39.06 78.13 156.25 312.5 625 1250 2500
pH 7.12 7.12 7.12 7.14 7.16 7.15 7.14 7.15 7.14 7.15
Osmolality mOsm 334 343 344 350 346 347 347 341 332 315

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test Procedure
Preliminary Cytotoxicity Test

Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. The preliminary cytotoxicity test was performed on cell cultures plated out at
1 x 10^7 cells/225 cm2 flask approximately 24 hours before dosing. This was demonstrated to
provide at least 20 x 10^6 available for dosing in each flask using a parallel flask, counted at the time of dosing. On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium (MEM). One flask per concentration was treated for 4-hours without metabolic activation and for 4-hours with metabolic activation (2% S9). The concentrations of test item used were 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL.

Exposure was for 4 hours at approximately 37 °C with a humidified atmosphere of 5% CO2 in air, after which the cultures were washed twice with phosphate buffered saline (PBS) before being detached from the flasks using trypsin. Cells from each flask were suspended in MEM with 10% FBS, a sample was removed from each dose group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm^2 flasks with 5 mL of MEM with 10% FBS and incubated for 6 to 7 days at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies (CE).

Results from the preliminary cytotoxicity test were used to select the test item concentrations for the mutagenicity experiment.

Mutagenicity Test – Main Experiment
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 1 x 10^7 cells/225 cm^2 flask approximately 24 hours being exposed to the test or control items. This was demonstrated to provide at least 20 x 10^6 available for dosing in each flask using a parallel flask. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with six test item concentrations, and vehicle and positive controls.

Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
The dose range of test item was 2.5 to 40 µg/mL in both the absence and presence of metabolic activation.

At the end of the treatment period the flasks were washed twice with PBS, detached from the flasks with trypsin and the cells suspended in MEM with 10% FBS. A sample of each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10^6 cells/flask in a 225 cm^2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm^2 flasks at 200 cells/flask to obtain the cloning efficiency, for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10% FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.

During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 and 5 to maintain logarithmic growth. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm^2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10^5 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 µg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.

The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.

Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.
Calculations
The cloning efficiency (CE), % control, mutant plate counts, mutant frequency/106 (MF10-6) and mutant frequency/106 survival rate (MFSV) were calculated using the following formulae:

CE% = ( x CE counts/200)x100
% Control = (CE% of Dose IDx/CE% of Dose ID0)x100

MF10-6 for each dose = Total mutant plate counts/2
MFSV for each dose = (MF10-6/CE)x100
Where:
Dose ID0 = Vehicle control values
Dose IDx = Concentration values

Small errors may occur when calculating mean cell concentrations and volumes for diluting; and in the calculation of means for cloning efficiency and mutant frequency; if these errors are ≤5% they are regarded to be within reasonable experimental error and considered not to affect the integrity of the study.

Evaluation criteria:

The following criteria will be used to determine a valid assay:
i) average absolute cloning efficiency of the Day 7 negative controls should exceed 50%. All assays below 50% cloning efficiency will be unacceptable.
ii) background (spontaneous) mutant frequency of the vehicle controls is generally within the historical range. The background values for the with and without activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays.
iii)concurrent positive controls should induce responses that are comparable with those generated in the historical positive control range and produce a statistically significant increase compared with the concurrent negative control.
iv)Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (20 mM, 2 mg/mL or 2 µL/mL whichever is lower, or 5 mg/mL for a UVCB*), or include the lowest precipitating concentration. Where a test item is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic concentration should be used. There is no maximum toxicity requirement for test items that are clearly mutagenic. Treatments that reduce relative clonal survival to <10% will not be scored for mutant frequency
v)Mutant frequencies are normally derived from sets of ten dishes/flasks for the mutant colony count and three dishes for cloning colony counts. To allow for contamination losses it is acceptable to score >8 mutant selection dishes and two cloning efficiency flasks.
vi)Five concentrations of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analyzed duplicate concentrations is considered necessary in order to accept a single assay for evaluation of the test item.

Statistics:

When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test

A dose range of 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL was used in the preliminary cytotoxicity test. The maximum dose tested was the maximum achievable concentration.

At the end of the exposure period, cloudy precipitate of the test item was observed at and above 39.06 µg/mL in both the absence and presence of metabolic activation. The precipitate became greasy / oily in appearance at and above 156.25 µg/mL in the absence of metabolic activation, and at and above 78.13 µg/mL in the presence of metabolic activation.

The results of the individual flask counts and their analysis are presented in Table 1. There was no evidence of any marked or dose related reductions in cloning efficiency in either the absence or presence of metabolic activation.

The maximum concentration selected for the main mutagenicity experiment was limited by the onset of test item precipitate as recommended by the OECD 476 guidelines.

Mutagenicity Test – Main Experiment
The concentrations of the controls and the test item are given in the table below:

Group Final concentration of GL500 (µg/mL)
4-hour without S9 0*, 2.5*, 5*, 10*, 20*, 30*, 40*, EMS 500* and 750*
4-hour with S9 (2%) 0*, 2.5*, 5*, 10*, 20*, 30*, 40*, DMBA 1.0* and 2.0*

In both the absence and presence of metabolic activation, all of the test item concentrations were plated for cloning efficiency and mutant frequency.

At the end of the exposure period, a cloudy precipitate of the test item was observed at 30 and 40 µg/mL in both the absence and presence of metabolic activation.

The Day 0 and Day 7 cloning efficiencies for the exposure groups in the absence and presence of metabolic activation are presented in Tables 2 and 3. There were no marked or dose-related reductions in in the Day 0 or 7 cloning efficiency values in either the absence or presence of metabolic activation.

The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 2 and Table 3. The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels in the main test, in either the absence or presence of metabolic activation

The vehicle control values were all considered to be within an acceptable range, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

* = Concentrations plated out for cloning efficiency and mutant frequency
EMS = Ethyl methane sulphonate
DMBA = Dimethyl benzanthracene

Remarks on result:

other: The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation.

Table 1 Preliminary Cytotoxicity Test Results

	4-hour -S9							4-hour +S9
Dose (µg/mL)	Count			Mean	% CE	% Control	Dose (µg/mL)	Count			Mean	% CE	% Control
0	168	174	162	168.0	84.0	100	0	185	170	171	175.3	87.7	100
9.77	127	138	127	130.7	65.3	78	9.77	154	143	140	145.7	72.8	83
19.53	141	145	141	142.3	71.2	85	19.53	152	156	171	159.7	79.8	91
39.06	156	162	153	157.0	78.5	93	39.06	157	139	151	149.0	74.5	85
78.13	148	184	164	165.3	82.7	98	78.13	163	159	164	162.0	81.0	92
156.25	163	174	154	163.7	81.8	97	156.25	127	136	130	131.0	65.5	75
312.5	157	155	169	160.3	80.2	95	312.5	149	157	141	149.0	74.5	85
625	150	169	148	155.7	77.8	93	625	157	138	152	149.0	74.5	85
1250	158	149	163	156.7	78.3	93	1250	162	144	142	149.3	74.7	85
2500	157	157	124	146.0	73.0	87	2500	141	124	145	136.7	68.3	78

Table 2 Main Experiment – 4-Hour Exposure without Metabolic Activation (S9)

		Day 0 Viability						Day 7 Viability						Day 7 Mutant
Dose(µg/mL)		Colonies/flask(200 cells plated/flask)			% CE	% Control	Mean % Control	Colonies/flask(200 cells plated/flask)			% CE	% Control	Mean % Control	Colonies/flask (2x105cells plated/flask)										MF	MFS 10-6	SD	Group MFS 10-6
0	A	161	170	154	80.8	100	100	174	150	149	78.8	100	100	3	5	3	3	5	3	2	1	1	0	13	16.5	2.03	17
0	B	142	136	137	69.2	100	100	190	170	176	89.3	100	100	2	3	0	7	3	4	3	0	7	2	15.5	17.4	2.03	17
2.5	A	162	180	171	85.5	105.8	113	144	155	127	71.0	90.1	86	4	4	3	2	3	4	4	3	2	2	15.5	21.8	1.42	19
2.5	B	165	157	175	82.8	119.8	113	145	166	133	74.0	82.8	86	2	6	5	2	1	2	1	2	1	1	11.5	15.5	1.42	19
5	A	149	174	158	80.2	99.2	102	141	120	127	64.7	82.0	76	2	0	0	2	3	2	4	4	1	1	9.5	14.7	1.80	18
5	B	162	144	132	73.0	105.5	102	126	135	110	61.8	69.2	76	1	6	4	2	3	0	1	6	2	1	13	21.0	1.80	18
10	A	156	145	159	76.7	94.8	101	121	126	129	62.7	79.5	84	4	3	2	2	0	4	1	5	1	0	11	17.6	1.57	14
10	B	151	135	157	73.8	106.7	101	155	177	142	79.0	88.4	84	1	0	1	2	2	1	4	2	0	4	8.5	10.8	1.57	14
20	A	164	161	162	81.2	100.4	98	155	143	143	73.5	93.2	98	2	5	4	2	2	2	2	2	3	1	12.5	17.0	1.24	15
20	B	147	120	128	65.8	95.2	98	198	186	168	92.0	103.0	98	3	2	5	3	2	0	3	1	3	3	12.5	13.6	1.24	15
30	A	156	159	166	80.2	99.2	107	151	143	147	73.5	93.2	83	2	3	7	3	5	4	5	4	4	3	20	27.2	1.71	22
30	B	166	152	157	79.2	114.5	107	124	144	119	64.5	72.2	83	2	1	5	3	2	3	0	4	1	1	11	17.1	1.71	22
40	A	142	147	129	69.7	86.2	86	153	147	151	75.2	95.3	93	0	5	1	4	0	0	1	2	2	1	8	10.6	1.50	10
40	B	128	106	124	59.7	86.3	86	170	158	153	80.2	89.7	93	2	0	1	4	1	3	0	1	0	2	7	8.7	1.50	10
EMS500	A	152	126	154	72.0	89.1	84	136	130	137	67.2	85.2	76	53	42	43	42	46	38	49	45	35	38	215.5	320.8	7.05	346
EMS500	B	95	120	115	55.0	79.5	84	118	114	127	59.8	67.0	76	30	49	39	58	35	50	43	48	53	40	222.5	371.9	7.05	346
EMS750	A	109	94	91	49.0	60.6	71	108	126	114	58.0	73.6	59	60	51	56	55	49	38	46	50	41	40	243	419.0	9.50	431
EMS750	B	114	105	120	56.5	81.7	71	79	75	83	39.5	44.2	59	35	39	29	38	36	43	32	40	23	35	175	443.0	9.50	431

Table 3 Main Experiment – 4-Hour Exposure with Metabolic Activation (2%S9)

		Day 0 Viability						Day 7 Viability						Day 7 Mutant
Dose (µg/mL)		Colonies/flask (200 cells plated/flask)			% CE	% Control	Mean % Control	Colonies/flask (200 cells plated/flask)			% CE	% Control	Mean % Control	Colonies/flask (2x105cells plated/flask)										MF	MFS 10-6	SD	Group MFS 10-6
0	A	149	161	160	78.3	100	100	162	163	148	78.8	100	100	2	1	2	3	0	1	1	1	1	1	6.5	8.2	1.42	12
0	B	173	171	178	87.0	100	100	163	157	133	75.5	100	100	2	2	1	3	4	2	1	3	0	6	12	15.9	1.42	12
2.5	A	155	155	148	76.3	97.4	103	135	133	122	65.0	82.5	100	1	3	0	3	2	1	3	0	1	0	7	10.8	1.61	14
2.5	B	192	194	182	94.7	108.8	103	179	177	173	88.2	116.8	100	6	2	3	2	1	2	5	3	4	2	15	17.0	1.61	14
5	A	136	160	151	74.5	95.1	106	152	159	128	73.2	92.8	96	3	3	1	2	2	1	3	4	2	2	11.5	15.7	1.13	12
5	B	208	201	204	102.2	117.5	106	146	144	163	75.5	100.0	96	1	1	1	0	2	2	0	1	0	3	5.5	7.3	1.13	12
10	A	144	171	145	76.7	97.9	97	136	139	137	68.7	87.1	92	0	3	1	0	2	1	2	1	1	3	7	10.2	1.77	15
10	B	177	189	137	83.8	96.4	97	142	141	153	72.7	96.2	92	7	2	5	2	1	2	5	3	2	1	15	20.6	1.77	15
20	A	165	165	163	82.2	104.9	114	126	119	112	59.5	75.5	90	3	1	1	1	1	1	1	2	1	2	7	11.8	0.97	13
20	B	220	210	210	106.7	122.6	114	154	157	159	78.3	103.8	90	4	1	1	1	3	2	3	3	1	2	10.5	13.4	0.97	13
30	A	175	174	180	88.2	112.6	103	154	166	145	77.5	98.3	95	4	2	1	0	3	3	3	2	5	2	12.5	16.1	1.48	19
30	B	146	153	185	80.7	92.7	103	137	134	141	68.7	90.9	95	3	5	4	5	1	1	3	1	3	4	15	21.8	1.48	19
40	A	151	168	190	84.8	108.3	96	153	143	136	72.0	91.3	105	3	2	2	1	3	1	0	2	2	1	8.5	11.8	1.92	18
40	B	143	157	138	73.0	83.9	96	182	186	170	89.7	118.8	105	5	5	5	5	7	5	1	4	3	5	22.5	25.1	1.92	18
DMBA 1	A	163	168	168	83.2	106.2	94	164	138	124	71.0	90.1	91	47	45	55	34	43	64	38	55	45	47	236.5	333.1	7.99	339
DMBA 1	B	135	147	147	71.5	82.2	94	133	136	148	69.5	92.1	91	57	52	61	40	49	50	36	42	47	46	240	345.3	7.99	339
DMBA 2	A	124	128	135	64.5	82.3	69	116	119	103	56.3	71.5	80	60	58	66	62	55	59	72	40	60	72	302	536.1	11.36	503
DMBA 2	B	95	95	104	49.0	56.3	69	138	116	144	66.3	87.9	80	79	76	71	54	66	56	31	60	64	67	312	470.4	11.36	503

DMBA = Dimethyl benzanthracene

CE = Cloning efficiency

MF = Mutant frequency

MFS = Mutant frequency per survivor

SD = Standard deviation

Conclusions:

Executive summary:

The purpose of this study is to assess the potential mutagenicity of a test item, supplied by the Sponsor, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

Methods

Chinese hamster (V79) cells were treated with the test item at six concentrations, in duplicate, together with vehicle (acetone) and positive controls in both the absence and presence of metabolic activation.

The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by the onset of test item precipitate as recommended by the OECD 476 guidelines. The concentrations of test item plated for cloning efficiency and expression of mutant colonies were as follows:

Exposure Group

Final concentration of GL500 (µg/mL)

4-hour without S9

2.5 , 5, 10, 20, 30, 40

4-hour with S9 (2%)

Results

The vehicle (acetone) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.

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.

The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels in the main test, in either the absence or presence of metabolic activation.

Conclusion

The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test item did not induce any increases in the percentage tail intensity or median percentage tail intensity in the liver or glandular stomach and therefore the test item was considered to be unable to induce DNA strand breakage to these tissues in vivo, under the conditions of the test.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Remarks:

Comet Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between10 October 2016 and 14 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was assigned Reliability 1 as the method is designed to be compatible with the procedures indicated in the OECD 489 Guideline (2014).

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2014

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Identification: GL500
Test Item (alternative names): GL500, GL520
Purity: 99.8%
CAS No.: 1571954-81-8
Action of test item: Plasticizer
Batch number: GLFG160607
Storage Conditions: At ambient temperature (10 to 30 ºC) in the dark (although may be used and formulated in light).
Purity/weighing factor: No adjustment for purity was made
Appearance: Clear colourless liquid
Expiry date: 07 June 2017

Species:

rat

Strain:

Wistar

Remarks:

HsdRCCHan™WIST

Sex:

male

Details on test animals or test system and environmental conditions:

Sufficient male Wistar Han™ (HsdRCCHan™WIST) rats were supplied by Envigo RMS (UK) Limited. At the start of the main test the males weighed 179.5 to 215.5 g, and were approximately eight to ten weeks old. Details of the individual animal weights, group means and standard deviations for the animals used in the main test are presented in Table 1. After a minimum acclimatization period of seven days the animals were selected at random and given a number unique within the study by tail marking and a number written on a color coded cage card.

The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Envigo Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

Arachis OIl

Details on exposure:

Experimental Design and Study Conduct

Test Item Preparation
For the purpose of this study the test item was freshly prepared as required as a solution at the appropriate concentration in arachis oil.
Determination by analysis of the concentration, homogeneity and stability of the test item preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline.

Positive Control Preparation
For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water (Laboratoire Aguettant Batch no. 3012083).

Vehicle Control
The Vehicle control (Arachis oil) was used as supplied.

Procedure
Range-finding Toxicity Test

A range-finding test was performed to find suitable dose levels of the test item following a double oral administration at zero and 24 hours. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

Groups of rats were dosed orally as follows:
Dose Level (mg/kg) Concentration (mg/mL) Dose Volume (mL/kg) Number of Rats
2000 200 10 2 male, 2 female

All animals were dosed twice 24 hours apart at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of the initial dosing.
Animals were observed 1 hour after each dosing and immediately prior to termination. Any deaths and evidence of overt toxicity were recorded at each observation.

Comet Test
Groups each of five males, were dosed twice with a 24 hour interval via the oral route with the test item at 2000, 1000 or 500 mg/kg. The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) approximately 4 hours following the second administration. In addition, two further groups of rats were included in the study; one group (five male rats) was dosed twice with a 24-hour interval via the oral route with the vehicle alone (arachis oil) and a second group (five male rats) was dosed twice orally with a 24-hour interval with N-Nitroso-N-methylurea (MNU) to act as the positive control. MNU is a positive control item that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test. The vehicle and positive control groups of rats were killed by humane euthanasia (carbon dioxide asphyxiation) 28 hours after the start of the test.

The experimental design is summarized as follows:

Treatment group Dose Level (mg/kg) Concentration (mg/mL) Dose volume (mL/kg) Kill Time (Hours After Initial Dosing) Animal Numbers
1. Vehicle Control (Arachis oil) 0 0 10 28 1 – 5
2. Positive Control (MNU) 25 2.5 10 28 6 – 10
3. GL500 2000 200 10 28 11 – 15
4. GL500 1000 100 10 28 16 – 20
5. GL500 500 50 10 28 20 – 25

All animals were observed for signs of overt toxicity and death one hour after each dosing and then immediately prior to termination.

Duration of treatment / exposure:

All animals were dosed twice 24 hours apart at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of the initial dosing.
Animals were observed 1 hour after each dosing and immediately prior to termination. Any deaths and evidence of overt toxicity were recorded at each observation.

Frequency of treatment:

See above

Dose / conc.:

500 other: mg/kg

Dose / conc.:

1 000 other: mg/kg

Dose / conc.:

2 000 other: mg/kg

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Positive control(s):

Identification: N-Nitroso-N-methylurea
Supplier: Astatec Inc
Batch Number: P102-01944
Purity: 90%
Expiry Date: 12 April 2017
Solvent: Water

Tissues and cell types examined:

Tissue Sample Requirements

Humane euthanasia was performed on the animals at the end of the exposure period, using a method that did not affect the integrity of the required tissues (carbon monoxide asphyxiation). Samples of liver and glandular stomach were obtained from each animal.

Sub-samples of the liver and glandular stomach were taken from the vehicle control animals and the dose group animals and preserved in 10% buffered formalin for possible histopathology investigations. Assessment of cytotoxicity by histopathology is conducted if the results from the Comet assay, or other observations, suggest cytotoxicity may be confounding the interpretation of the Comet assay.

Details of tissue and slide preparation:

Tissue Preparation

Liver - A small piece of liver was excised (approximately 1 cm^3) and washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension.

Glandular Stomach – The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for possible histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension was obtained by further scraping of the exposed tissue.

The above procedures were performed under subdued lighting and the Comet Assay tissues/cells were processed as quickly as possible using ice-cold buffers to maintain the tissues and cell preparations at low temperature

Slide Preparation

Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature prior to the start of the experiment. Prior to use in the study the slides were labelled for animal number, project number and tissue type.

Once the cell suspensions had been obtained, approximately 30 µL was added to 270 µL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 µL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension was immediately covered with a glass coverslip and kept at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify. All of the slides went through the subsequent processing.

Once the LMP agarose had set the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight.

After the lysis phase had been completed the slides were removed from the lysing solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis unit, which was filled with chilled electrophoresis buffer, until the slide surface was just covered. The slides were then left for approximately 20 minutes to allow the DNA to unwind. When the DNA unwinding period had finished the slides were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for approximately 20 minutes. The buffer in the bath was maintained at low temperature (approximately 2-10 °C) during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis. The voltage and current at the start and end of the electrophoresis period was recorded. The aim was to induce sufficient migration of the DNA so that minimal sized Comets are produced in the nuclei of vehicle control cells.

At the end of the electrophoresis period the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer (0.4M Tris pH 7.5) and allowed to rest for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer performed twice. The slides were then carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry.

Once dry the slides were stored prior to scoring. Two of the four processed slide gels were scored and the remaining slides were stored as backup slides.

Evaluation criteria:

Acceptability Criteria

The following criteria will be used to determine a valid assay:

• The concurrent negative control is comparable with the laboratory historical negative control range.
• The positive controls induce responses that are comparable with those in the laboratory positive control range.
• Adequate numbers of cells and doses have been analysed.
• The highest dose level selected meets the requirements of the guideline and the study plan

Statistics:

When a less than clear response is observed a comparison will be made between the vehicle control groups and each corresponding treatment dose group on the percentage tail intensity data using individual slide scored values.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Range-Finding ToxicityTest

A range-finding test was performed to find suitable dose levels of the test item following oral administration at zero and 24 hours. The data are summarized as follows:

Dose Level (mg/kg)	Sex	Number of Animals Treated	Route	Deaths on Day		Total Deaths
Dose Level (mg/kg)	Sex	Number of Animals Treated	Route	0	1	Total Deaths
2000	Male	2	oral	0	0	0/2
2000	Female	2	oral	0	0	0/2

In animals dosed with test item there were no premature deaths, and no clinical signs observed after dosing or at termination.

Based on the above data the maximum recommended dose (MRD) of the test item,2000 mg/kg, was selected for use in the main test, with 1000 and 500 mg/kg selected as the lower dose levels. There was no noticeable difference in clinical signs between the male and female animals and therefore only male animals were used for the main test. Due to the absence of any toxicity at the maximum recommended dose level the groups for the main test were limited to five animals per group.

Comet Assay

Mortality Data and ClinicalObservations

There were no premature deaths or clinical signs seen in any of the test item dose groups during the main test.

Evaluation of Comet AssaySlides

A summary of the results for each of the tissues of the Comet Assay, liver and glandular stomach, is given in Table 1.

The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control range. The positive control item (MNU) produced a marked increase in the percentage tail intensity and median percentage tail intensity in the liver and glandular stomach. The test method itself was therefore operating as expected and was considered to be valid under the conditions of the test.

There was no marked increase in percentage tail intensity for any of the test item dose levels in the glandular stomach or liver tissues when compared to the vehicle control, confirming the test item did not induce DNA damage in the liver or glandular stomach.

There was no marked increase in hedgehog frequency for any of the test item dose levels in any of the tissues investigated.

Table 1 Summary of Results for Percentage Tail Intensity,MedianPercentageTail Intensity and Percentage Hedgehogs for EachTissue

Glandular Stomach

Dose Level	Group Mean % Hedgehogs	Group Mean % Tail Intensity	Group Mean of Mean of Median % Tail Intensity per Animal
Vehicle	5.27	7.41	5.22
500 mg/kg	6.55	6.37	4.03
1000 mg/kg	5.15	4.51	2.74
2000 mg/kg	6.04	4.99	3.28
Positive (MNU)	6.95	40.70	38.85

Liver

Dose Level	Group Mean % Hedgehogs	Group Mean % Tail Intensity	Group Mean of Mean of Median % Tail Intensity per Animal
Vehicle	0.68	0.37	0.01
500 mg/kg	0.30	0.35	0.01
1000 mg/kg	0.39	0.27	0
2000 mg/kg	0.40	0.35	0.01
Positive (MNU)	0.29	26.67	26.67

Conclusions:

Executive summary:

The Comet Assay has been designed using the recommendations of the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington DC 1999, as described by Ticeet al., 2000. The method is designed to be compatible with the procedures indicated in the OECD 489 Guideline (2014).

The primary target tissues of this assay were liver and glandular stomach.

Methods

A range-finding test was performed to find suitable dose levels of the test item and to determine the appropriate sex for the main test. The Comet assay main test was conducted at the maximum recommended dose (MRD) 2000 mg/kg with 1000 and 500 mg/kg as the lower dose levels, using five male animals per group. Animals were killed 4 hours after the second dose administration, the glandular stomach and liver tissues processed. Comet slides were then prepared and processed prior to scoring.

Further groups of rats were given a double oral dose of arachis oil (five rats) or N-Nitroso-N- methylurea (five rats), to serve as vehicle and positive controls respectively.

Results

The test item was tested up to the maximum recommended dose level of 2000 mg/kg using male animals only. There was no evidence of an increase in the percentage tail intensity or median percentage intensity in animals dosed with the test item dose groups when compared to the concurrent vehicle control group.

The positive control material produced a marked increase in the percentage tail intensity and median percentage tail intensity in both the liver and glandular stomach, indicating that the test method was working as expected.

Conclusion

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

All in-vivo and invitro studies were concluded with a negative result therefore according to the CLP criteria the test item GL500 will be considered as non hazardous with respect to the genetic toxicity endpoint.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TAlOO		TA1535		*WP2uvrA*		TA98		TA1537
82		13		17		21		4
80	(81)	20	(18)	20	(21)	19	(17)	4	(5)
82		21		27		12		8

Test Period		From: 05 July 2013				I		To: 08 July 2013
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-oair substitution strains						Frameshift strains
		TAlOO		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	74 103 96	(91) 15.1#	16 13 16	(15) 1.7	21 32 29	(27) 5.7	19 37 16	(24) 11.4	9 (11) 7 5.3 17
	1.5 µg	87 104	(94) 8.7	12 13	(15) 4.4	29 25	(30) 5.6	25 17	(20) 4.2	7 (12) 13 4.6
		92		20		36		19		16
	5µg	102 96	(97) 4.2	13 19	(16) 3.1	27 27	(28) 2.3	21 28	(24) 3.5	7 (12) 19 6.1
		94		15		31		24		11
	15 µg	91 90 88	(90) 1.5	13 9 17	(13) 4.0	12 27 33	(24) 10.8	23 21 37	(27) 8.7	11 (14) 12 4.4 19
	50 µg	79 87 82	(83) 4.0	16 20	(18) 2.1	33 24	(29) 4.5	17 21	(19) 2.0	5 (8) 11 3.1
		79 87 82	(83) 4.0	17		29		19		7
	150 µg	92 90	(93) 3.1	11 13	(12) 1.2	23 32	(27) 4.5	20 25 28	(24) 4.0	13 (14) 19 4.2
		96		11		27		20 25 28	(24) 4.0	11
	500 µg	94 115 95	(101) 11.8	11 11	(14) 4.6	25 27	(26) 1.2	13 23	(20) 6.4	13 (15) 8 7.6
		94 115 95	(101) 11.8	19		27		25		23
	1500 µg	95P 95P	(90) 9.2	16P 15P	(15) 0.6	31P 32P	(29) 4.9	15 P 19P	(15) 3.5	19P (21) 21P 2.0
		79P		15 P		23 P		12 P		23 P
	5000 µg	78P 94P 95P	(89) 9.5	15P 12P 8P	(12) 3.5	17P 17P 20P	(18) 1.7	24P 20P 35P	(26) 7.8	19P (15) 9P 5.3 17 P
Positive controls S9-Mix (+)	Name DoseLevel No. ofRevertants	2AA		2AA		2AA		BP		2AA
		1 µg		2µg		10 µg		5µg		2 µg
		732 625	(739) 117.1	160 100 95	(118) 36.2	250 215	(230) 17.9	138 115	(123) 13.0	138 (135) 132 3.1
		859		160 100 95	(118) 36.2	226		116		136

Registration Dossier

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Diss Factsheets

Reaction mass of bis(2-ethylhexyl) terephthalate, butyl 2-ethylhexyl terephthalate, and dibutyl terephthalate

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

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Reference 1

Methods

Results

Conclusion

Reference 2

Reference 3

Endpoint conclusion

Genetic toxicity in vivo

Description of key information

Link to relevant study records

Reference

Endpoint conclusion

Additional information

Justification for classification or non-classification

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