2-ethylhexyl 10-ethyl-4,4-dimethyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (Stankowski, 1996)

In vitro Gene mutation (Reverse mutation assay/Ames): S. typhimurium and E. coli- negative in all strains with and without metabolic activation.

 

HPRT Study (Rashmi, 2019)

Under the conditions of this study, the test material is considered as weakly mutagenic at the concentration of 0.0625 µL/mL, both in the presence and absence of metabolic activation.

 

Chromosome Aberration (Rashmi, 2019)

Under the conditions of the study the test material is considered as non-clastogenic up to the concentration of 0.125 µL/mL, both in the presence and absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Preferred study for this SIDS endpoint; EPA/OECD GLP compliance was noted. Composition/purity of the test substance not reported.

Qualifier:

according to guideline

Guideline:

other: OECD, USEPA and USFDA and proposed revisions to OECD (1994). Specific guidelines not provided.

Deviations:

yes

Remarks:

, analyses were not performed to verify the homogeneity, stability or accuracy of preparation of the test and control article dosing solutions.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Strains TA1535 and TA100 detect base pair substitution mutations affecting the hisG46 allele.
Strain TA98 detects frameshift mutations affecting the hisD3052.
Strain TA1537 detects frameshift mutations affecting the hisC3076 allele.
Strain TA102 can detect a variety of genetic damage affecting AT base pairs in the hisG428 allele.
Strain WP2 uvrA detects AT base pair mutations at the trp locus.

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver homogenate

Test concentrations with justification for top dose:

16.7, 50, 167, 500, 1670, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance demonstrated solubility in the solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Evaluated in the absence of S9: sodium azide; 9-aminoacridine; 2-nitrofluorene; mitomycin C; ENNG. In the presence of S9: 2-Aminofluorene and 2-anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation) and preincubation.
Evaluated in both toxicity prescreen and mutation assays using both the liquid pre-incubation and plate incorporation treatment.


DURATION
- Preincubation period: 30 minutes (liquid pre-incubation method)
- Exposure duration: 48-hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 30 minutes
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours


SELECTION AGENT (mutation assays): Na2HPO4

NUMBER OF REPLICATIONS: triplicate

Evaluation criteria:

A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the solvent control value. If the test article does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine or tryptophan-independent revertants.

Statistics:

Statistical analyses were conducted using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. A positive result was defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan-independent revertants, with at least one dose level inducing a revertant frequency that was two-fold the solvent control value. An equivocal result was declared if the test substance did not induce a statistically significant, dose-dependent increase in revertant frequency, but did induce a revertant frequency at one dose level that was two-fold the spontaneous control value. A negative result was defined as the absence of a statistically significant or dose-dependent increase in the number of histidine- or tryptophan-independent revertants. Statistical analyses were only conducted when a 50% increase in revertant frequency (relative to the concurrent negative controls) was observed.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Primary test results revealed that in the liquid pre-incubation assay, growth was inhibited in strains TA1535, TA1537, and TA100 at >= 500 ug/plate (in the presence and absence of metabolic activation. Toxicity to strain TA98 was reported at >=1670 ug/plate (without activation) and >=500 ug/plate (with activation). Growth was inhibited in strain TA102 at >= 1670 ug/plate (both with and without activation). A dose level of 5000 ug/plate inhibited growth of strain WP2 uvrA without metabolic activation, and >=1670 ug/plate with metabolic activation. In the plate incorporation assay, both with and without metabolic activation, growth was inhibited in strain TA1537 at >=500 ug/plate and in strain TA1535 at 5000 ug/plate. In the presence of S9, growth in TA100 and TA102 was inhibited at 5000 ug/plate, and without S9 at >=1670 ug/plate. A dose level of >=500 ug/plate inhibited growth in TA98 (without S9) and at >=1670 ug/plate (with S9). No growth inhibition was observed in WP2 uvra. Again, the test substance was reported to be incompletely soluble at levels >= 500 ug/plate.


RANGE-FINDING/SCREENING STUDIES: Preliminary test results revealed that the test substance produced inhibited growth in both Salmonella tester strains (TA1537 and TA100) at doses >= 500 ug/plate, under liquid pre-incubation conditions. Additionally, the test substance was found to be incompletely soluble at levels >= 500 ug/plate.


COMPARISON WITH HISTORICAL CONTROL DATA: all positive and negative control values in both assays were within acceptable historical ranges.

Remarks on result:

other: At dose levels below the solubility of the test substance

Conclusions:

Interpretation of results: Negative

The results of the tests conducted on the test substance, for both liquid pre-incubation and plate incorporation treatments, in the presence and absence of a metabolic activation system, were negative at dose levels below the solubility of the test substance.

Executive summary:

The test article was evaluated in the Ames/Salmonella-E. coli Reverse Mutation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium (TA1535, TA1537, TA100, TA98, TA102) and at the tryptophan locus in one Escherichia coli tester strain (WP2 uvrA), in both the presence and absence of an exogenous metabolic activation system (S9).

Toxicity of the test article was first evaluated in a preliminary toxicity screen using both liquid pre-incubation and plate incorporation treatment conditions. Duplicate cultures were treated at doses of 50.0, 167, 500, 1670 and 5000 ug/plate, and the DMSO solvent control, in the absence of S9. Results of the prescreen indicated that the test substance produced inhibited growth in both Salmonella tester strains at doses >/=500 ug/plate under liquid pre-incubation conditions. In addition, the test article was found to be incompletely soluble at doses >/=500 ug/plate.

The test article next was evaluated for mutagenicity using both treatment conditions. Based upon the results of the prescreen, the test substance was evaluated in triplicate cultures in all six tester strains at doses of 16.7, 50.0, 167, 500, 1670 and 5000 ug/plate with and without S9. Six doses of the test substance were evaluated in the event of unacceptable toxicity and/or insolubility at the highest dose levels evaluated in the mutation assay. The S9 mixture included 6% (v/v) Aroclor 1254 -induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors. Except for strain WP2 uvrA under plate incorporation conditions, inhibited growth again was observed for all strains/S9/treatment combinations at the highest 1 -3 doses evaluated. In addition, the test article again was found to be incompletely soluble at doses >/= 500 ug/plate. Revertant frequencies for all doses of the test substance in all tester strains with and without S9, under both treatment conditions, approximated or were less than those observed in the concurrent negative control cultures. All positive and negative control values in both assays were within acceptable ranges.

Therefore, the results for the test substance were negative in the Ames/Salmonella-E. coli Reverse Mutation Assay, using liquid pre-incubation and plate incorporation treatments, under the test conditions.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 July 2018 to 20 April 2019

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:

29 July 2016

Deviations:

no

Principles of method if other than guideline:

Dose formulation analysis for the dose formulations was not be done as per the sponsor requirement.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: CHO AA8 cells, Batch No. 5000062 procured from American Type Culture Collection (ATCC) was used for the test.
- Suitability of cells: The derivative of the CHO-K1, CHO AA8 Cells were used as the test system as recommended in the guideline OECD 476.
- Preparation of Cultures: A frozen cryovial of cells was thawed immediately in the water bath. Cells were transferred to a sterile flask with culture medium containing 10 % Foetal Bovine Serum (FBS) with antibiotics (1 % penicillin and streptomycin) and incubated at 37 ± 1 °C and 5 ± 1 % CO2 for 3 days. The cell lines were trypsinised and the trypsinised cultures were sub-cultured before use in the experiment. Approximately 2 × 10^6 cells per culture flask (initial cytotoxicity test and gene mutation test) were seeded using culture medium with 10 % FBS with antibiotics (1 % penicillin and streptomycin). Four additional flasks were seeded and kept for incubation along with flasks for treatment to determine cell count at the beginning of the treatment to determine the adjusted Cloning Efficiency. The flasks were incubated at 37 ± 1 °C with 5 ± 1 % CO2 for 22 hours and 40 minutes (initial cytotoxicity test) to 23 hours (gene mutation test).

MEDIA USED
- Type and identity of media: Alpha Minimal Essential Medium (MEM) without ribonucleosides containing 10% FBS and antibiotics (1% penicillin and streptomycin) were used. The pH of the culture medium was 7.33 to 7.37. The media was stored at 2 to 8 °C until use and thawed at room temperature before use.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes. Cells free of mycoplasma were used for the experiment.
- Periodically 'cleansed' against high spontaneous background: Yes. The cultures were cleansed of pre-existing mutant cells by culturing in HAT Medium and then returned to normal growth medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Based on the results of solubility, pH and precipitation tests an initial cytotoxicity test was conducted at the concentrations of 0.0625, 0.125, 0.25, 0.50, 1 and 2 µL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation. At a concentration of 0.0625 µL/mL, the Relative Survival was in the range 10 to 20 %. Therefore 0.0625 µL/mL was selected as the highest concentration for testing in the gene mutation tests.
Based on the results of the initial cytotoxicity test, the gene mutation test was conducted at the concentrations of 0.0078, 0.0156, 0.0312, and 0.0625 µL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: A solubility test was conducted to determine the maximum concentration or workable suspension of the test material in the vehicle compatible with the test system up to the maximum concentration of 200 µL/mL. DMSO was used to prepare the stock solution and dilutions of the test material. The test material was dissolved separately in a small amount of DMSO. A solubility test was conducted in distilled water and DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

INITIAL CYTOTOXICITY TEST
- Test Procedure
For tests with exogenous metabolic activation, 1 mL of S9 mix was added to all the flasks. A volume of 100 µL of vehicle/different concentrations of test material was added to four plates per culture to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test material for 3 hours and 15 minutes at 37 ± 1 °C with 5 ± 1 % CO2.
For tests without exogenous metabolic activation, a volume of 100 µL of vehicle/different concentrations of test material was added to four plates per culture to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test material for 3 hours 45 minutes at 37 ± 1 °C with 5 ± 1 % CO2.
Post incubation period (Set 1 and 2 - with and without metabolic activation, respectively), medium from each flask was aspirated and monolayer was washed with DPBS. Cells were trypsinised. Trypsinisation was stopped by adding culture media followed by collecting the media with cells. The duration of treatment was 3 hours 45 minutes.
Four plate treatments were collected in pre-labelled tubes and centrifuged at 800 rpm for 10 minutes. Supernatant was discarded and cell pellet was retained. Each treatment replicate was plated in triplicate with a cell concentration of 200 cells/5 mL media in cell culture 25 cm² dishes and incubated at 37 ± 1 °C with 5 ± 1 % CO2 for 8 - 9 days.
After the incubation period, medium from each culture flask was aspirated and stained with 5 % Giemsa stain. The number of colonies formed was counted manually.

- Determination of Cytotoxicity
The Cytotoxicity level was determined using the following formulae:
Adjusted Cloning Efficiency (ACE) = CE × (No. of cells at the end of treatment / No. of Cells at the beginning of the treatment)
Relative Survival (RS) = (ACE (Treated) / ACE (Vehicle Control)) × 100
Cloning efficiency (CE) is the percentage of cells plated at a low density that are able to grow into a colony that can be counted.

GENE MUTATION TEST
- Criteria for Selection of Concentrations for Gene Mutation Test
Since the test material showed no precipitation and no change in pH up to 2 µL/mL, the same concentration was considered as the highest concentration in the initial cytotoxicity test. At a concentration of 0.0625 µL/mL, the Relative Survival was in the range 10 to 20 %. Therefore 0.0625 µL/mL was selected as the highest concentration for testing in the gene mutation tests. Four concentrations i.e. 0.0078, 0.0156, 0.0312 and 0.0625 µL/mL were selected for gene mutation test, based on initial cytotoxicity test. As the response at 0.0625 µL/mL was not clearly negative, gene mutation test II was conducted to examine the reproducibility of the findings.

- Test Procedure for Gene Mutation Test
The gene mutation test was carried out as described for the initial cytotoxicity test. Each treatment group was maintained with four plate cultures. Cells were exposed to the test material for 3 hours and 35 minutes both with exogenous metabolic activation and without exogenous metabolic activation respectively in the gene mutation test at 37 ± 1 °C with 5 ± 1 % CO2.
For Set 1 and 2 (with and without metabolic activation, respectively), the duration for gene mutation and gene mutation II was 3 hours and 35 minutes and 3 hours and 49 minutes, respectively.
Four plate treatments were pooled into a pre-labelled tube and centrifuged at 800 rpm for 10 minutes. Supernatant was discarded and cell pellet was retained.
Cytotoxicity was determined as described above.

- Expression of Mutant Phenotype
The replicate cultures were sub-cultured in duplicates at a density of 1 × 10^6 cells/culture flask. Cells were incubated at 37 ± 1 °C with 5 ± 1 % CO2, followed by sub-culturing with an interval of 3 days for the remaining 9 days of expression period.

- Selection of Mutant Phenotypes and Plating for Cloning Efficiency
Following the 9 day expression period, each replicate treatment culture was pooled and sub-cultured in quintuplicates at a density of 4 × 10^5 cells per group with culture media containing 10 µM of 6-thioguanine and 200 cells /dish in triplicates without 6-thioguanine for determination of cloning efficiency. Dishes were incubated at 37 ± 1 °C with 5 ± 1 % CO2 for 7 days. After the incubation period, medium from each dish was aspirated and stained with 5 % Giemsa stain and the number of colonies formed were counted manually.

- Data Presentation
Adjusted Cloning Efficiency and Relative Survival were calculated using the formulae described above. Mutant Frequency of each treatment was calculated using the following formula:
Mutant Frequency (MF) = Cloning efficiency of mutant colonies in selective medium / Cloning efficiency in non-selective medium
Cloning efficiency = Number of colonies / Number of cells plated.
MF is expressed as mutants per 2 × 10^6 clonable cells.

ACCEPTABILITY CRITERIA
Acceptance of a test is based on the following criteria:
- The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guideline 476.
- Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control.
- Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
- Adequate number of cells and concentrations are analysable (according to OECD guideline 476).
- The criteria for the selection of top concentration are consistent with those described in OECD guideline 476.

Evaluation criteria:

INTERPRETATION OF RESULTS
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- The increase is concentration-related when evaluated with an appropriate trend test;
- Any of the results are outside the distribution of the historical negative/vehicle control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
A test chemical is considered clearly negative if, in all experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- There is no concentration-related increase when evaluated with an appropriate trend test;
- All results are inside the distribution of the historical negative/vehicle control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive as described above, performing a repeat experiment possibly using modified experimental conditions will be considered in consultation with the sponsor.

Statistics:

Data of mutant frequencies was analysed using SPSS Software version 22 for differences among vehicle control, treatment and positive control groups using ANOVA following Dunnett’s test and ANOVA following Tukey (trend test).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

SOLUBILITY, PRECIPITATION AND pH TEST
The test material was miscible in DMSO at 200 µL/mL. There was no change in pH and moderate precipitate was observed at and up to 2 µL/mL in culture medium. On the basis of the results, 2 µL/mL was selected as the highest concentration for the initial cytotoxicity test.

INITIAL CYTOTOXICITY TEST
The results of the initial cytotoxicity test indicated that the Relative Survival is greater than 10 % at ≤ 0.0625 µL/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Above this level, survival rate decreased to below 5 % observed at 0.125, 0.25, 0.50, 1 and 2 µL/mL after test material exposure.

GENE MUTATION TEST I
In the gene mutation test I, the cells were treated with test material at the concentrations of 0.0078, 0.0156, 0.0312 and 0.0625 µL/mL using DMSO as the vehicle in four plate cultures both in the presence of metabolic activation and absence of metabolic activation.
The test material resulted in mutant frequencies of 17.11 to 36.59 per 2 × 10^6 cells in the presence of metabolic activation with 23.17 per 2 × 10^6 cells in the vehicle control. In the absence of metabolic activation, mutant frequencies of 18.67 to 36.96 per 2 × 10^6 cells were observed with 23.75 per 2 × 10^6 cells in the vehicle control. Only in the presence of S9, did the increase in mutant frequency achieve statistical significance when compared to the concurrent vehicle control (p<0.05). However, neither in the absence nor presence of S9 was there a concentration-related increase when evaluated with an appropriate trend test. Furthermore, the mutant frequencies at the highest concentration tested (0.0625 µL/mL; 36.59 per 2 × 10^6 cells in the presence of metabolic activation and 36.96 per 2 × 10^6 cells in the absence of S9) exceeded the distribution of the historical vehicle control data (26.46 per 2 × 10^6 cells) or 26.74 per 2 × 10^6 cells, in the presence or absence of S9 respectively).
There was no evidence of excessive cytotoxicity (˂10 % RS) at any of the concentrations in both presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 22.62 to 79.76 % and in the absence of metabolic activation the RS values ranged from 21.69 to 80.72 % respectively.
The positive control, 3 µg/mL of benzo(a)pyrene, in the presence of metabolic activation, resulted in a RS value of 77.38 % and mutant frequency of 260.00 per 2 × 10^6 cells which was statistically significant when compared with the vehicle control.
The positive control, 1 µg/mL of 4-nitroquinoline N-oxide, in the absence of metabolic activation, resulted in a RS value of 75.90 % and mutant frequency of 260.53 per 2 × 10^6 cells and was statistically significant when compared with that of vehicle control.
As the response at 0.0625 µL/mL was not clearly negative, gene mutation test II was conducted to examine the reproducibility of the findings.

GENE MUTATION TEST II
In gene mutation test II, the test material, at 0.0625 µL/mL resulted in mutant frequencies of 41.46 per 2 × 10^6 cells in the presence of metabolic activation with 22.09 per 2 × 10^6 cells in the vehicle control. In the absence of metabolic activation, mutant frequencies of 43.90 per 2 × 10^6 cells were observed with 22.89 per 2 × 10^6 cells in the vehicle control. There was a statistical significant increase in the mutant frequency at the tested concentration (0.0625 µL/mL, with and without S9) when compared with the vehicle control and the observed mutant frequencies exceeded the 95 % confidence level of the laboratory’s negative historical control data.

DISCUSSION
In the gene mutation test I, there was no statistically significant increase in the number of mutant colonies observed when compared with vehicle control at any of the tested concentrations except in highest concentration 0.0625 µL/mL (+S9) which showed statistical significance and there was no concentration-related increase when evaluated with an appropriate trend test. However, the mutant frequencies at the highest concentration tested (0.0625 µL/mL) exceeded the distribution of the historical vehicle control data. The gene mutation test I met two criteria out of three and hence could not be considered clearly negative.
In the gene mutation test II, there was a statistically significant increase in the number of mutant colonies observed at 0.0625 µL/mL treated both presence and absence of metabolic activation when compared with vehicle control. The mutant frequencies at the highest concentration tested (0.0625 µL/mL) exceeded the distribution of the historical vehicle control data. Hence, the test concentration 0.0625 µL/mL is considered positive as the test met the all the positive criteria. Based on the results obtained, the test material is considered as weakly mutagenic at the concentration of 0.0625 µL/mL both in the presence and absence of metabolic activation under the tested laboratory conditions.

Remarks on result:

other: The test material is considered as weakly mutagenic at the concentration of 0.0625 µL/mL

Table 1: Summary of Parallel Cytotoxicity Test - Gene Mutation Test I

Set No.	Treatment	Concentration (µL/mL)	Average colony count ± SD			*Cloning Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set 1 +S9	Vehicle Control (DMSO)	-	164.67	±	4.51	0.82	0.84	-
	Test Material	0.0078	143.33	±	3.79	0.72	0.67	79.76
		0.0156	109.00	±	6.56	0.55	0.51	60.71
		0.0312	78.00	±	2.00	0.39	0.34	40.48
		0.0625	46.00	±	3.61	0.23	0.19	22.62
	Benzo(a)pyrene	0.003	145.67	±	6.03	0.73	0.65	77.38
Set 2 -S9	Vehicle Control (DMSO)	-	163.33	±	5.13	0.82	0.83	-
	Test Material	0.0078	135.00	±	3.00	0.68	0.67	80.72
		0.0156	112.33	±	9.29	0.56	0.51	61.45
		0.0312	80.00		5.00	0.40	0.34	40.96
		0.0625	43.33	±	3.79	0.22	0.18	21.69
	4 Nitroquinoline N-oxide	0.001	138.67	±	6.51	0.69	0.63	75.90

*Note: Cloning Efficiency = 200 cells plated for each replicate

 RS = (Adjusted CE in treated culture/Adjusted CE in the vehicle control) x 100

 CE = Number of colonies/Number of cells plated

Adjusted CE = CE x (Number of cells at the end of treatment/number of cells at the beginning of treatment)

 

Table 2: Summary of Parallel Cytotoxicity Test - Gene Mutation Test II

Set No.	Treatment	Concentration (µL/mL)	Average colony count ± SD			*Cloning Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set 1 +S9	Vehicle Control (DMSO)	-	168.33	±	6.51	0.84	0.86	-
	Test Material	0.0625	53.67	±	5.69	0.27	0.24	27.91
	Benzo(a)pyrene	0.003	145.33	±	4.51	0.73	0.60	69.77
Set 2 -S9	Vehicle Control (DMSO)	-	164.33	±	4.51	0.82	0.86	-
	Test Material	0.0625	53.00	±	6	0.27	0.23	26.74
	4 Nitroquinoline N-oxide	0.001	142.67	±	3.51	0.71	0.60	69.77

*Note: Cloning Efficiency = 200 cells plated for each replicate

RS = (Adjusted CE in treated culture/Adjusted CE in the vehicle control) x 100

CE = Number of colonies/Number of cells plated

Adjusted CE = CE x (Number of cells at the end of treatment/number of cells at the beginning of treatment)

 

Table 3: Summary of Gene Mutation Test I

Set No.	Treatment	Concentration (µL/mL)	**Average colony count ± SD			Cloning Efficiency in selective media	Cloning Efficiency in non-selective media	Average Mutant Colonies/ 2 x 10^6 cells	Mutant Frequency/ 2 x 10^6 cells	Sig. (p value)
Set 1 +S9	Vehicle Control (DMSO)	-	163.00	±	2.65	0.0000095	0.82	19	23.17	-
	Test Material	0.0078	152.33	±	7.51	0.0000065	0.76	13	17.11	0.278
		0.0156	128.33	±	6.66	0.0000065	0.64	13	20.31	0.922
		0.0312	109.67	±	6.81	0.0000065	0.55	13	23.64	1.000
		0.0625	82.33	±	5.86	0.0000075	0.41	15	36.59*	0.041
	Benzo(a)pyrene	0.003	150.67	±	4.16	0.0000975	0.75	195	260.00*	0.000
Set 2 -S9	Vehicle Control (DMSO)	-	160.00	±	5.00	0.0000095	0.80	19	23.75	-
	Test Material	0.0078	150.67	±	4.93	0.0000070	0.75	14	18.67	0.501
		0.0156	137.00	±	2.65	0.0000070	0.69	14	20.29	0.729
		0.0312	120.00	±	1.00	0.0000075	0.60	15	25.00	0.999
		0.0625	91.33	±	3.21	0.0000085	0.46	17	36.96	0.066
	4 Nitroquinoline N-oxide	0.001	152.67	±	3.21	0.0000990	0.76	198	260.53*	0.000

**Note: Cloning efficiency = 200 cells plated for each replicate

*Statistically significant (p˂0.05)              

Mutant Frequency = Cloning efficiency of mutant colonies in selective medium / Cloning efficiency in non-selective medium

Table 4: Summary of Gene Mutation Test II

Set No.	Treatment	Concentration (µL/mL)	**Average colony count ± SD			Cloning Efficiency in selective media	Cloning Efficiency in non-selective media	Average Mutant Colonies/ 2 x 10^6 cells	Mutant Frequency/ 2 x 10^6 cells	Sig. (p value)
Set 1 +S9	Vehicle Control (DMSO)	-	172.33	±	8.50	0.0000095	0.86	19	22.09	-
	Test Material	0.0625	81.67	±	5.51	0.0000085	0.41	17	41.46*	0.007
	Benzo(a)pyrene	0.003	142.67	±	3.06	0.0000895	0.71	179	252.11*	0.000
Set 2 -S9	Vehicle Control (DMSO)	-	166.00	±	3.61	0.0000095	0.83	19	22.89	-
	Test Material	0.0625	82.33	±	3.51	0.0000090	0.41	18	43.90*	0.012
	4 Nitroquinoline N-oxide	0.001	142.00	±	9.17	0.0000940	0.71	188	264.79*	0.000

**Note: Cloning efficiency = 200 cells plated for each replicate

*Statistically significant (p˂0.05)

Mutant Frequency = Cloning efficiency of mutant colonies in selective medium / Cloning efficiency in non-selective medium 

Conclusions:

Under the conditions of this study, the test material is considered as weakly mutagenic at the concentration of 0.0625 µL/mL, both in the presence and absence of metabolic activation.

Executive summary:

The potential for the test material to cause gene mutation in CHO AA8 cells at the HPRT locus was investigated in accordance with the standardised guideline OECD 476 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was found to be miscible in DMSO at 200 µL/mL. There was no change in pH and moderate precipitate was observed at and up to 2 µL/mL in culture medium. Based on the results of the solubility, pH and precipitation tests an initial cytotoxicity test was conducted at the concentrations of 0.0625, 0.125, 0.25, 0.50, 1 and 2 µL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 to 6 hours).

The results of the initial cytotoxicity test indicated that the Relative Survival is greater than 10 % at concentrations ≤ 0.0625 µL/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on the result 0.0625 µL/mL was selected as the highest concentration for the gene mutation test. The gene mutation test was conducted at the concentrations of 0.0078, 0.0156, 0.0312, and 0.0625 µL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours 35 minutes).

Benzo(a)pyrene and 4 nitroquinoline N-oxide were used as positive controls for the gene mutation test.

Cytotoxicity was assessed by determining the adjusted Cloning Efficiency and Relative Survival in the test. 

In gene mutation test I, a small increase in mutant frequency (MF) was observed at the highest concentration (0.0625 µL/mL) in the absence and presence of S9, which exceeded the laboratory’s 95 % historical control data range. However, only in the presence of S9, did the increase in MF achieve statistical significance when compared to the concurrent vehicle control (p<0.05). Although the MF observed in the absence and presence of S9 increased with increasing concentration, there was no statistically significant linear trend.

As the response at 0.0625 µL/mL was not clearly positive, gene mutation test II was conducted to examine the reproducibility of the findings. There was a statistically significant increase in the mutant frequency at the tested concentration (0.0625 µL/mL) in the presence and absence of S9 when compared with the vehicle control. Furthermore, these MF exceeded the laboratory’s 95 % historical control data range.

In both tests, positive controls resulted in mutant frequencies, which were statistically significant when compared with the vehicle control.

Under the conditions of this study, the test material is considered as weakly mutagenic at the concentration of 0.0625 µL/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 July 2018 to 27 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human Peripheral Blood Lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
Human peripheral lymphocytes was collected from the blood of healthy, young, non-smoking (male) donors 29 and 28 years of age with no known recent exposure to genotoxic chemicals or radiation. Blood from each individual was collected in sodium heparin vacutainer and analysed using Advia 2120. As all the parameters were in acceptable range, blood was used from a single donor for the initial cytotoxicity and chromosomal aberration test.
- Modal number of chromosomes: The chromosome number of human lymphocytes is 2n=46. Since fixation procedures often result in the breakage of a proportion of metaphase cells with loss of chromosomes, cells with 46 ± 2 number of chromosomes were considered for analysis.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Culture Media: RPMI Media supplemented with 10% FBS and antibiotics (1 % Penicillin-Streptomycin). The pH of the culture medium used was 7.28 to 7.26. The media was stored at 2 to 8 °C till use and was thawed to room temperature before use.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix metabolic activation

Test concentrations with justification for top dose:

The following set of criteria was followed for the selection of concentrations for chromosomal aberration test.
• Three analysable concentrations were used for chromosomal aberration test.
• Based on the results of cytotoxicity test, the concentrations selected for the chromosomal aberration test were 0.0312, 0.0156 and 0.0625 µL/mL of the test material as low, mid and high concentrations respectively.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was conducted to determine the maximum concentration or workable suspension solution of the test material in the vehicle compatible with the test system at 200 µL/mL and the test material was soluble in dimethyl sulphoxide at 200 µL/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

DURATION
- Exposure duration: 3 to 6 hours and 20 to 24 hours
- The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, the cells were treated with a metaphase-arresting substance (colchicine), harvested, stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.

NUMBER OF REPLICATIONS: Three slides were prepared for each treatment replicate.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- Slide Preparation: Pellets were mixed with 3 to 4 mL of freshly prepared warm 0.56 % potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 3 to 4 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cell suspension was incubated for 10 minutes at room temperature and later suspension was centrifuged at 2 200 rpm for 10 minutes. The procedure was repeated twice by adding 3 mL of cold acetic acid: methanol fixative (1:3).
Clean slides were stored in a beaker with distilled water and kept in the refrigerator for at least 1 hour before use. The cell suspension was mixed using a pipette and a few drops of the suspension were aspirated and dropped onto the chilled slide pre labeled with study number, with (+S9) or without metabolic activation(-S9), treatment/group and slide number. The slides were air dried.
A minimum of 3 slides were prepared for each treatment replicate. Slides were stained using 5 % Giemsa stain for 15 minutes.
- Slide Evaluation: Coding of slides was not carried out for the initial cytotoxicity test. For each replicate minimum of 500 cells were scored.
Percent mitotic index (MI %) was determined by the following formula:
Mitotic Index

MI% = (Number of Mitotic cells / Total number of cells scored) × 100

Percent reduction in mitotic index was obtained by using the formula:

= [(Percentage MI of VC - Percentage MI of treated)/Percentage MI of VC] ×100

VC: Vehicle Control
MI: Mitotic Index

- All slides including vehicle control, treatment and positive controls of chromosomal aberration test were coded before evaluation in the main test.
- Cytotoxicity was determined by calculating percentage reduction in mitotic index (%).
- Concurrent measures of mitotic index for all treated and vehicle control cultures were determined.
- The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate and the metaphases with aberrations were recorded in raw data.
- Gaps were recorded separately and reported but not included in the total aberration frequency.


DETERMINATION OF CYTOTOXICITY
- Based on the results of solubility, precipitation and pH tests, an initial cytotoxicity test was conducted for the selection of test concentrations for the chromosomal aberration test. The concentrations selected for initial cytotoxicity test were 0.125, 0.25, 0.5, 0.25, 1 and 2 µL/mL.
- Initial Cytotoxicity Test Procedure:
Whole blood of volume 0.5 mL was added to each tube containing culture media of volume 4.5 mL supplemented with 2 % phytohaemagglutinin (PHA) and incubated for 44 to 48 hours at 37±1 °C and 5 ± 1 % CO2.
Post 44 to 48 hours of incubation with PHA, cells from tubes were centrifuged at 1500 rpm for 10 minutes, supernatant was discarded.
Cell pellet was re-suspended with 2 to 3 mL fresh media.
The treatments in the initial cytotoxicity test were:
Set 1 (+S9): vehicle control and test material: 4 h and 4 minutes.
Set 2: (-S9): vehicle control and test material: 4 h and 4 minutes.
Set 3: (-S9): vehicle control and test material: 21 h and 42 minutes.
For tubes with metabolic activation (+S9) - set 1, cell suspension was mixed with 50 µL each of the respective test concentrations/vehicle, 0.5 mL of S9 mix and volume was made up to 5 mL with culture media.
For tubes without metabolic activation (-S9) - set 2 and 3, cell suspension was mixed with 50 µL each of the respective test concentrations/vehicle and the volume was made up to 5 mL with culture media.
All the test material concentrations and controls were maintained in duplicate.
Cells were incubated both with metabolic activation (+S9) - set 1 for 3 to 6 hours, without metabolic activation (-S9) - set 2 for 3 to 6 hours and without metabolic activation (-S9) for 20 to 24 hours set 3 at 37 ± 1 °C and 5 ± 1 % CO2.
The treatment for set 1 and 2 tubes were terminated post 3 to 6 hours of incubation, by centrifugation at 1500 rpm for 10 minutes.
Supernatant was discarded and cell pellet was mixed with 5 mL of culture medium and incubated further to complete 20 to 24 hours starting from the start of treatment.
1 hour 48 mins (in initial cytotoxicity test) and 1 hour 23 minutes (in chromososmal aberration test) prior to harvesting, colchicine of concentration 0.3 µg/mL was added to all the tubes of set 1, 2 and 3. Post incubation of 1 to 3 hours with colchicine, cell suspension was collected to pre-labelled tubes and centrifuged for 10 minutes at 1500 rpm.
The final concentration of vehicle did not exceed 1 % for organic solvent.


- OTHER: INTERPRETATION OF RESULTS
Providing that all acceptability criteria are fulfilled, a test material was considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test material concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
- The increase is dose-related when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical negative control data.
The test material is then considered to be able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test material was considered to be clearly negative if, in all experimental conditions examined:
- None of the test material concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
- There is no concentration-related increase when evaluated with an appropriate trend test.
- All results are inside the distribution of the historical vehicle control data.
The test material is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.

An increase in the number of polyploid cells may indicate that the test material has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations.
An increase in the number of cells with endoreduplicated chromosomes may indicate that the test material has the potential to inhibit cell cycle progression.

Rationale for test conditions:

The in vitro chromosomal aberration test employs primary cell cultures derived from healthy human donors. The primary cell cultures of human whole blood were selected on the basis of growth ability in culture, stability of the karyotype. This provides the opportunity to test using the same test system which the in vitro test is predictive of in vivo genotoxic events. Further as per the regulatory requirements the human peripheral blood lymphocytes is one of the recommended test system.

Evaluation criteria:

Study was accepted if;
- The concurrent vehicle control is within the 95 % control limits of the distribution of the laboratory’s historical negative/vehicle control database.
- Concurrent positive controls produced a statistically significant increase compared with the concurrent vehicle control and positive controls should induce responses that are compatible with those generated in the historical positive control data base.
- Adequate number of cells (at least 300 well spread metaphases per concentration) and concentrations (at least three analysable concentrations) were analysed.

Statistics:

Data (Percentage of cells with aberrations) were analysed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test material groups using ANOVA following Dunnett’s test and ANOVA following Tukey (trend test) at a 95 % level of confidence (p < 0.05) and the statistical significance was designated by the superscripts in the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group.

Species / strain:

lymphocytes: Human Peripheral Blood Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- In the chromosomal aberration test, the cells were treated with the test material at the concentrations of 0.0312, 0.0625 and 0.125 µL/mL using DMSO as the vehicle in duplicate for short term (3 to 6 hours) both in the presence and absence of metabolic activation. Similarly for long term (20 to 24 hours) in the absence of metabolic activation.
- There was no statistically significant increase in the number of aberrant cells in test material treated cultures when compared with vehicle control at any of the concentration levels tested and there was no concentration-related increase when evaluated with an appropriate trend test. The reduction in MI observed at 0.125 µL/mL was 43.55 % in the presence of metabolic activation and 44.17 % in the absence of metabolic activation for short term treatments. Similarly, the reduction in MI observed at 0.125 µL/mL was 45.03 % in the absence of metabolic activation system for long term treatment.
- The observed mean percent aberrant cells at 0.0312, 0.0625 and 0.125 µL/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) were 1.00, 1.00 and 0.34 respectively. Similarly, the observed mean percent aberrant cells at 0.0312, 0.0625 and 0.125 µL/mL in the absence of metabolic activation (short term treatment 3 to 6 hours) were 0.67, 1.00 and 0.67 respectively.
- The observed mean percent aberrant cells at 0.0312, 0.0625 and 0.125 µL/mL in the absence of metabolic activation, long term (20 to 24 hours) were 1.00, 0.34 and 1.33 respectively. There was no cells with endoreduplication chromosomes and no polyploidy cells were observed.

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test material was miscible in dimethyl sulphoxide at 200 µL/mL.
- Precipitation: A precipitation test was conducted at 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 23 hours and 50 minutes of incubation, no change in pH and no precipitation was observed at any of the concentration tested. Hence, 2 µL/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.125, 0.25, 0.5 and 1 µL/mL of test material.

CONTROL DATA
- Positive control data:
Positive control, 10 µg/mL of cyclophosphamide monohydrate, in the presence of metabolic activation (3 to 6 hours), induced 8.67 % of aberrated cells which was statistically significant compared to the vehicle control (1.0 %). The reduction in mitotic index was 4.30 % when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of mitomycin-C, in the absence of metabolic activation (3 to 6 hours), induced 9.0 % of aberrated cells which was statistically significant to the vehicle control (1.33 %). The reduction in mitotic index observed was 7.94 % when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of mitomycin-C, in the absence of metabolic activation (20 to 24 hours), induced 8.67 % of aberrated cells which was statistically significant when compared to the vehicle control (0.67 %). The reduction in mitotic index observed was 7.98 % when compared with the vehicle control for long term treatment. The concurrent vehicle control values were within the 95 % control limits of the distribution of the laboratory’s historical vehicle control database.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Initial cytotoxicity test:
Treatment of cultures with the test material at the concentrations of 0.125, 0.25, 0.5, 1 and 2 µL/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) showed reduction in mitotic index and the observed values were 47.25 %, 79.75 %, 95.53 %, 100 % and 100 % respectively. In the absence of metabolic activation (short term treatment 3 to 6 hours) the obtained reduction in mitotic index was 45.56 %, 77.08 %, 97.11 %, 100 % and 100 % respectively. In the absence of metabolic activation (long term treatment 20 to 24 hours) the obtained reduction in mitotic index was 48.79 %, 87.40 %, 95.77 %, 100 % and 100 % respectively.

Conclusions:

Under the conditions of the study the test material is considered as non-clastogenic up to the concentration of 0.125 µL/mL, both in the presence and absence of metabolic activation.

Executive summary:

The test material was evaluated for chromosomal aberrations in human lymphocytes, according to the standardised guideline OECD 473 and under GLP conditions.

The test material was miscible in dimethyl sulphoxide at 200 µL/mL. A precipitation test was conducted at 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 23 hours and 50 minutes of incubation, no change in pH and no precipitation was observed at any of the concentration tested. Hence, 2 µL/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.125, 0.25, 0.5 and 1 µL/mL of test material.

In a pre-study, the percentage reduction in Mitotic Index was in the range of 45.56 to 97.05 at 0.125, 0.25 and 0.5 µL/mL. At 1 and 2 µL/mL, the percentage reduction in Mitotic Index was 100. As the percentage reduction in MI was not more than 45 ± 5 % at 0.125 mg/mL, the same concentration was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.0312 and 0.0625 µL/mL.

In the chromosomal aberration test, the cells were treated with the test material at the concentrations of 0.0312, 0.0625 and 0.125 µL/mL using DMSO as the vehicle. The treatment was carried out in duplicate for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide monohydrate (+S9 for short term) at the concentration of 10 µg/mL and mytomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.

The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, the cells were treated with a metaphase-arresting substance (colchicine), harvested, stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.

There was no statistically significant increase in the number of aberrant cells in test material treated cells when compared with vehicle control and there was no concentration-related increase when evaluated with an appropriate trend test. The reduction in MI observed at 0.125 µL/mLwas 43.55 % in the presence of metabolic activation and 44.17 % in the absence of metabolic activation for short term treatments. Similarly, the reduction in MI observed at 0.125 µL/mL was 45.03 % in the absence of metabolic activation system for long term treatment.  

The observed mean percent aberrant cells at 0.0312, 0.0625 and 0.125 µL/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) were 1.00, 1.00 and 0.34 respectively. Similarly, the observed mean percent aberrant cells at 0.0312, 0.0625 and 0.125 µL/mL in the absence of metabolic activation (short term treatment 3 to 6 hours) were 0.67, 1.00 and 0.67 respectively.

The observed mean percent aberrant cells at 0.0312, 0.0625 and 0.125 µL/mL in the absence of metabolic activation, long term (20 to 24 hours) were 1.00, 0.34 and 1.33 respectively.

The cultures treated with positive controls for the short-term period (3 to 6 hours) both in the presence and absence of metabolic activation, and for the long-term period (20 to 24 hours) in the absence of metabolic activation induced were 8.67 %, 9.00 % and 8.67 % of aberrant cells respectively, which was statistically significant compared with the respective vehicle control. This demonstrated sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate. The concurrent vehicle control values were within the 95 % control limits of the distribution of the laboratory’s historical vehicle control database.

Under the conditions of the study the test material is considered as non-clastogenic up to the concentration of 0.125 µL/mL, both in the presence and absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

It is proposed that a comet assay (in liver and duodenum) combined with bone marrow micronucleus evaluation is an appropriate and scientifically justified assay for in vivo evaluation of the genotoxicity hazard identified for DMTE in vitro.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vivo mammalian somatic cell study: combined gene mutuation and cytogenicity / micronucleus assay

Type of information:

experimental study planned

Study period:

To be determined upon receipt of ECHA final decision

Justification for type of information:

Please see attached justification

We have received from ECHA a draft decision on this testing proposal via REACH-IT on 30 March 2022. The communication number is TPE-D-2114591210-58-01/D

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

In vitro

Ames Test (Stankowski, 1996)

Data from an OECD 471 equivalent study are used to address gene mutation in bacterial cells. Salmonella typhimurium strains TA1537, TA1535, TA98, TA100 and TA102 and Escherichia coli strain WP2 uvrA were tested in the presence and absence of metabolic activation. The test substance was evaluated using both the liquid pre-incubation and plate incorporation treatment methods. Revertant frequencies for all doses in all strains with and without activation, under both treatment conditions, approximated or were less than those observed in the concurrent negative control cultures. The result for the test substance is negative.

 

HPRT Study (Rashmi, 2019)

The potential for the test material to cause gene mutation in CHO AA8 cells at the HPRT locus was investigated in accordance with the standardised guideline OECD 476 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was found to be miscible in DMSO at 200 µL/mL. There was no change in pH and moderate precipitate was observed at and up to 2 µL/mL in culture medium. Based on the results of the solubility, pH and precipitation tests an initial cytotoxicity test was conducted at the concentrations of 0.0625, 0.125, 0.25, 0.50, 1 and 2 µL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 to 6 hours).

The results of the initial cytotoxicity test indicated that the Relative Survival is greater than 10 % at concentrations ≤ 0.0625 µL/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on the result 0.0625 µL/mL was selected as the highest concentration for the gene mutation test. The gene mutation test was conducted at the concentrations of 0.0078, 0.0156, 0.0312, and 0.0625 µL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours 35 minutes).

Benzo(a)pyrene and 4 nitroquinoline N-oxide were used as positive controls for the gene mutation test.

Cytotoxicity was assessed by determining the adjusted Cloning Efficiency and Relative Survival in the test. 

In gene mutation test I, a small increase in mutant frequency (MF) was observed at the highest concentration (0.0625 µL/mL) in the absence and presence of S9, which exceeded the laboratory’s 95 % historical control data range. However, only in the presence of S9, did the increase in MF achieve statistical significance when compared to the concurrent vehicle control (p<0.05). Although the MF observed in the absence and presence of S9 increased with increasing concentration, there was no statistically significant linear trend.

As the response at 0.0625 µL/mL was not clearly positive, gene mutation test II was conducted to examine the reproducibility of the findings. There was a statistically significant increase in the mutant frequency at the tested concentration (0.0625 µL/mL) in the presence and absence of S9 when compared with the vehicle control. Furthermore, these MF exceeded the laboratory’s 95 % historical control data range.

In both tests, positive controls resulted in mutant frequencies, which were statistically significant when compared with the vehicle control.

Under the conditions of this study, the test material is considered as weakly mutagenic at the concentration of 0.0625 µL/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

 

Chromosomal Aberration (Rashmi, 2019)

The test material was evaluated for chromosomal aberrations in human lymphocytes, according to OECD test guideline and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was miscible in dimethyl sulphoxide at 200 µL/mL. Precipitation test was conducted at 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 23 hours and 50 minutes of incubation, no change in pH and no precipitation was observed at any of the concentration tested. Hence, 2 µL/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.125, 0.25, 0.5 and 1 µL/mL of test item.

In a pre-study, the percentage reduction in Mitotic Index was in the range of 45.56 to 97.05 at 0.125, 0.25 and 0.5 µL/mL. At 1 and 2 µL/mL, the percentage reduction in Mitotic Index was 100. As the percentage reduction in MI was not more than 45 ± 5 % at 0.125 mg/mL, same has been selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.0312 and 0.0625 µL/mL.

In the chromosomal aberration test, the cells were treated with the test material at the concentrations of 0.0312, 0.0625 and 0.125 µL/mL using DMSO as the vehicle. The treatment was carried out in duplicate for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mytomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.

The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, the cells were treated with a metaphase-arresting substance (colchicine), harvested, stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.

There was no statistically significant increase in the number of aberrant cells in test material treated cells when compared with vehicle control and there was no concentration-related increase when evaluated with an appropriate trend test. The reduction in MI observed at 0.125 µL/mLwas 43.55 % in the presence of metabolic activation and 44.17 % in the absence of metabolic activation for short term treatments. Similarly, the reduction in MI observed at 0.125 µL/mL was 45.03 % in the absence of metabolic activation system for long term treatment.  

The observed mean percent aberrant cells at 0.0312, 0.0625 and 0.125 µL/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) were 1.00, 1.00 and 0.34 respectively. Similarly, the observed mean percent aberrant cells at 0.0312, 0.0625 and 0.125 µL/mL in the absence of metabolic activation (short term treatment 3 to 6 hours) were 0.67, 1.00 and 0.67 respectively.

The observed mean percent aberrant cells at 0.0312, 0.0625 and 0.125 µL/mL in the absence of metabolic activation, long term (20 to 24 hours) were 1.00, 0.34 and 1.33 respectively.

The cultures treated with positive controls for the short-term period (3 to 6 hours) both in the presence and absence of metabolic activation, and for the long-term period (20 to 24 hours) in the absence of metabolic activation induced were 8.67 %, 9.00 % and 8.67 % of aberrant cells respectively, which was statistically significant compared with the respective vehicle control. This demonstrated sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate. The concurrent vehicle control values were within the 95 % control limits of the distribution of the laboratory’s historical vehicle control database.

Under the conditions of the study the test material is considered as non-clastogenic up to the concentration of 0.125 µL/mL, both in the presence and absence of metabolic activation.

 

In vivo

There is currently no available in vivo genotoxicity information for DMTE. 

The registrant is proposing to carry out an in vivo mutagenicity study on DMTE itself; it is proposed that a comet assay (in liver and duodenum) combined with bone marrow micronucleus evaluation is an appropriate and scientifically justified assay for in vivo evaluation of the genotoxicity hazard identified for DMTE in vitro.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.