Ethylene bis(3-mercaptopropionate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 15, 2022 - July 21, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted 29th July, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S9 Fraction

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time
without S9 mix: 37, 111.1, 333.3 and 1000 μg/mL test item
with S9 mix: 74.1, 222.2, 666.6 and 2000 μg/mL test item

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 7.9, 15.7, 31.3 and 62.5 μg/mL test item

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 7.9, 15.7, 31.3 and 62.5 μg/mL test item

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 74.1, 222.2, 666.6 and 2000 μg/mL test item

(selected on the basis of a pre-test on cytotoxicity)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 5E05 cells/dish
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
- Experiment A: 3-hour treatment, harvest 20 hours from the beginning of treatment
- Experiment B: 20-hour treatment, harvest 20 hours from the beginning of treatment / 20 (without S9 mix)- and 3-hour (with S9 mix) treatment, harvest 28 hours
from the beginning of treatment

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: Colchicine (0.2 μg/mL) 2.5-3 hours prior to harvesting
- Methods of slide preparation and staining technique used including the stain used:
Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.
300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration as well as the negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
Additionally, the number of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- pretest for selection of concentrations: Relative Increase in Cell Counts (RICC)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Chromatid and chromosome type aberrations (gaps, deletions and exchanges), the number of polyploid and endoreduplicated cells

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, the test item 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 laboratory historical negative control data.

Both biological and statistical significance are considered together.
There is no requirement for verification of a clearly positive or negative response.

Statistics:

For statistical analysis the CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The data were checked for a linear trend in number of cells with aberrations (without gaps). with treatment dose using the adequate regression analysis by Microsoft
Excel software.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality.
Clear cytotoxicity in the range required by the guideline (55 ± 5 %) was observed at the highest concentrations without metabolic activation in Experiment A and experiment B (between 52 and 54%). In experiment A and in experiment B at the highest recommended concentration with metabolic activation the observed cytotoxicity was between 41 and 45%.

Conclusions:

In conclusion, GDMP did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to the cytotoxic concentrations in the absence and up to the maximum recommended concentration in the presence of metabolic activation.
Thus, the test item is considered as being non-clastogenic in this system.

Executive summary:

The test item GDMP, dissolved in DMSO (Dimethyl sulfoxide), was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:
Experiment A with 3/20 h treatment/sampling time
without S9 mix: 37, 111.1, 333.3 and 1000 μg/mL test item
with S9 mix: 74.1, 222.2, 666.6 and 2000 μg/mL test item

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 7.9, 15.7, 31.3 and 62.5 μg/mL test item

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 7.9, 15.7, 31.3 and 62.5 μg/mL test item

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 74.1, 222.2, 666.6 and 2000 μg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).
No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in osmolality after treatment with the test item. In the pre-test on cytotoxicity in the presence of metabolic activation dose related decrease (in the physiological range) was observed in the pH values. In the main experiments the pH at concentrations of 666.6 and 2000 μg/mL were lower than the concurrent control value. However, these values were in the physiological range Clear cytotoxicity in the range required by the guideline (55 ± 5 %) was observed at the highest concentrations without metabolic activation in Experiment A and experiment B (between 52 and 54%). In experiment A and in experiment B at the highest recommended concentration with metabolic activation the observed cytotoxicity was between 41 and 45%.
In experiment A, no increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed, neither in the absence nor in the presence of metabolic activation. All values for aberrant cells were within the historical control range of 2-5 aberrant cells excluding gaps.
In experiment B no increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed in the absence (20-hour treatment/28-hour sampling time) nor in the presence of metabolic activation. All values for aberrant cells were within the historical control range of 2-5 aberrant cells excluding gaps.

There were no polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.
The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, GDMP did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to the cytotoxic concentrations in the absence and up to the maximum recommended concentration in the presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Justification for type of information:

Currently, do data on toxicokinetics/metabolism is available for this category. Based on structural features (e.g. sterical hindrance) it is however assumed, that ester cleavage would not be fast and complete, especially since the substances contain up to 6 ester functions, which are in addition sterically shielded. Therefore, it seems more reasonable to base the category hypothesis on structural similarity.
In addition, it is not clear yet, whether the strength of the effects vary in a predictable manner, or if no relevant variations occur. However, there are variations in structure (number of ester bonds and consequently number of free -SH groups) and physicochemical properties (especially water solubility and log Kow). It is assumed that these variations will also be reflected by variations in effect levels. Therefore, scenario 4 is the working hypothesis for the time being.
More data points within the category are needed to further strengthen the category hypothesis. The scenario selection will be re-evaluated after the studies are finished.
This currently selected scenario covers the category approach for which the read-across hypothesis is based on structural similarity. For the REACH information requirement under consideration, the property investigated in studies conducted with different source substances is used to predict the property that would be observed in a study with the target substance if it were to be conducted. Similar properties are observed for the different source substances; this may include absence of effects for every member of the category.
There are expected to be differences in strength of the effects forming a regular pattern. The prediction will be based on a worst-case approach. The read-across is a category approach based on the hypothesis that the substances in this category share structural similarities with common functional groups. This approach serves to use existing data on acute toxicity, repeated-dose toxicity, and reproductive toxicity endpoints for substances in this category.
The hypothesis corresponds to Scenario 4 of the RAAF. The substances GDMP, TMPMP, PETMP, and Di-PETMP are esters of a common acid, 3-mercaptopropionic acid (3-MPA). The key functionality of the substances within this category is the presence of free SH-groups. It is hypothesised that the strength of effects correlates with the number of SH-groups. In addition, differences in bioavailability are expected to influence the strength of effects.
For details, please refer to the category document attached to Iuclid section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without metabolic activation: >= 40µg/ml with metabolic activation: >= 313µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

PETMP did not show genotoxicity in the mammalian gene mutation test. Based on the categroy approach GDMP is considered as non genotoxic in the mammalian gene mutation test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-04-23 to 2012-05-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Liver post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254. S9 was collected from 20 - 30 rats.

Test concentrations with justification for top dose:

5000 µg GDMP/plate based on cytotoxicity (scarce background lawn and reduction of the number of revertants)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48-72 h

SELECTION AGENT (mutation assays): his-free medium

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1: Plate incorporation test -without metabolic activation

S9 Mix	Conc. (µg/ plate)	Number of revertants (mean number of colonies per plate, n=3)
		TA98	TA100	TA1535	TA1537	TA1537	WP2 uvr A
–	0	40.7	157.3	22.0	5.0	5.7	29.3
–	31.6	32.0	179.3	19.0	4.3	4.7	54.3
–	100	37.7	140.0	19.3	2.3	3.3	53.0
–	316	37.3	168.0	14.0	5.0	4.3	51.7
–	1000	29.3	171.3	20.7	2.3	3.3	49.3
–	3160	27.3	140.7	18.0	5.3	5.3	43.7
–	5000	10.3#	87.0#	9.3#	1.0#	2.0#	12.7#
Pos controls ‑S9	Name	2-NF	NaN3	NaN3	9-AA	2-NF	NQO
	Conc. (µg/plate)	10	10	10	100	10	5
	No. of revertants per plate	134.7	865.7	118.0	140.7	174.3	249.3

2-NF   2-Nitrofluorene

NQO   4-Nitroquinoline-1-oxide

9-AA   9-Aminoacridine

NaN₃   Sodium azide

#        scarce background lawn

 

Table 2: Plate incorporation test -with metabolic activation

S9 Mix	Conc. (µg/ plate)	Number of revertants (mean number of colonies per plate, n=3)
		TA98	TA100	TA1535	TA1537	TA1537	WP2 uvr A
+	0	45.0	177.0	15.7	6.7	5.3	54.3
+	31.6	37.7	163.3	16.0	4.7	6.7	42.3
+	100	39.7	153.7	14.0	2.0	6.3	52.7
+	316	35.3	161.0	19.7	5.0	5.3	50.7
+	1000	26.7	166.3	22.3	1.7	4.3	53.0
+	3160	35.3	161.3	18.0	3.7	4.3	52.0
+	5000	15.0#	91.3#	7.7#	1.0#	1.7#	16.3#
Pos controls +S9	Name	BaP	NaN3	NaN3	9-AA	2-NF	NQO
	Conc. (µg/plate)	5	2	2	5	5	2
	No. of revertants per plate	120.7	869.0	125.7	142.7	170.3	211.0

BaP    Benzo[a]pyrene

2-AA   2-Aminoanthracene

#        scarce background lawn

 

 

 

Table 3: Pre-incubation test -without metabolic activation

S9 Mix	Conc. (µg/ plate)	Number of revertants (mean number of colonies per plate, n=3)
		TA98	TA100	TA1535	TA1537	TA1537	WP2 uvr A
–	0	35.3	153.7	17.7	4.7	5.7	47.0
–	31.6	33.3	153.7	16.0	6.3	5.3	47.7
–	100	23.7	155.0	15.7	5.0	5.3	51.3
–	316	35.7	1296.7	17.7	5.3	5.0	51.7
–	1000	24.7	137.0	26.7	5.7	6.3	48.0
–	3160	4.07	14137	22.7	5.0	5.7	43.7
–	5000	9.3#	59.0#	4.3#	1.0#	1.7#	9.3#
Pos controls ‑S9	Name	2-NF	NaN3	NaN3	9-AA	2-NF	NQO
	Conc. (µg/plate)	10	10	10	100	10	5
	No. of revertants per plate	175.7	945.3	127.7	127.0	230.1	224.7

2-NF   2-Nitrofluorene

NQO   4-Nitroquinoline-1-oxide

9-AA   9-Aminoacridine

NaN₃   Sodium azide

#        scarce background lawn

 

Table 4: Pre-incubation test -with metabolic activation

S9 Mix	Conc. (µg/ plate)	Number of revertants (mean number of colonies per plate, n=3)
		TA98	TA100	TA1535	TA1537	TA1537	WP2 uvr A
+	0	36.7	161.7	16.7	2.7	4.7	56.0
+	31.6	27.7	148.3	19.7	6.0	7.0	39.0
+	100	28.0	141.0	18.7	4.7	5.0	45.7
+	316	35.3	163.7	11.7	5.7	4.7	30.7
+	1000	23.7	152.0	19.7	4.7	4.7	50.3
+	3160	22.7	168.7	15.0	3.0	5.3	46.3
+	5000	10.7#	43.7#	6.7#	1.0#	1.7#	8.3#
Pos controls +S9	Name	BaP	NaN3	NaN3	9-AA	2-NF	NQO
	Conc. (µg/plate)	5	2	2	5	5	2
	No. of revertants per plate	178.0	980.7	173.3	113.0	227.7	255.3

BaP    Benzo[a]pyrene

2-AA   2-Aminoanthracene

#        scarce background lawn

 

Conclusions:

GDMP is negative in the Ames test, with and without metabolic activation.

Executive summary:

GDMP was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and in the Escherichia coli strain WP2 uvr A in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.
GDMP was completely dissolved in dimethylsulfoxide (DMSO). The vehicle DMSO served as the negative control.

Preliminary test
GDMP was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 μg/plate were tested. Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 5000 μg GDMP/plate.
Hence, 5000 μg GDMP/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test, respectively.

Main study
Six concentrations ranging from 31.6 to 5000 μg GDMP/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 5000 μg GDMP/plate, in all Salmonella typhimurium strains and in the Escherichia coli strain WP2 uvr A.

No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for GDMP, tested up to a cytotoxic concentration of 5000 μg/plate, in the Salmonella typhimurium and in the Escherichia coli test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The substances GDMP, TMPMP, PETMP, and Di-PETMP are esters of a common acid, 3-mercaptopropionic acid (3-MPA). All category members share the same mercaptopropionic acid moiety with two to 6 free SH group per MPA unit. The MPA unit with free SH is a prerequisite for this category. A justification for read-across is attached to Iuclid section 13.

 

Bacterial reverse mutation assay

Negative Ames tests are available for GDMP and PETMP. 

 

GDMP was examined for iits mutagenic potential in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and Escherichia coli WP2 uvr A in two independent experiments, each carried out without and with metabolic activation. The first experiment was carried out as a plate incorporation test and the second as a preincubation test.
Cytotoxicity was noted at the top concentration of 5000 μg GDMP/plate. No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for GDMP, tested up to a cytotoxic concentration of 5000 μg/plate, in the Salmonella typhimurium and in the Escherichia coli test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

 

The genotoxic potential of the test item PETMP was assessed in a Bacterial reverse mutation assay according to OECD Guideline 471 and EU method B13/14. Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with solutions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A white, cloudy precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.

 

Chromosome aberration assay

Negative chromosomal aberration assays are available for GDMP and PETMP.

 

The test item GDMP, dissolved in DMSO (Dimethyl sulfoxide), was tested in a chromosome aberration assay in V79 cells in two independent experiments in accordance with the current OECD Guideline 473. For the cytogenetic experiments the concentrations were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation).
Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).
Clear cytotoxicity in the range required by the guideline (55 ± 5 %) was observed at the highest concentrations with and without metabolic activation in both experiments.
In both experiments, no increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed, neither in the absence nor in the presence of metabolic activation. All values for aberrant cells were within the historical control range of 2-5 aberrant cells excluding gaps.
There were no polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.
In conclusion, GDMP did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to the cytotoxic concentrations in the absence and up to the maximum recommended concentration in the presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

 

To investigate the potential of PETMP to induce structural chromosome aberrations in Chinese hamster V79 cells in the presence and absence of metabolic activation, an in vitro chromosome aberration assay according to OECD TG  473 was carried out. PETMP was tested up to cytotoxic concentrations.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations.

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all concentrations treated with the test item were within the historical control data of the negative control.

In experiment I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item PETMP did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.

Therefore, the test item is considered to be non-clastogenic.

 

Mammalian cell gene mutation assay

A negative mouse lymphoma assay is available for PETMP.

 

The test item PETMP was assessed for its potential to induce mutations at the thymidine kinase locus using the mouse lymphoma cell line L5178Y with and without metabolic activation in accordance with OECD Guideline 476.

No biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item PETMP is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cellline L5178Y when tested up to cytotoxic concentrations.

 

 

Conclusion

The entirety of available genotoxicity data in combination with the absence of structural alerts for genotoxicity in this category will be used to strengthen the weight of the evidence for non-genotoxicity.

 

Justification for classification or non-classification

GDMP was negative in an Ames test, with and without metabolic activation. PETMP showed negative results in cytogenicitiy and gene mutation study in mammalian cells. Therefore GDMP is considered to not show genotoxicity. A further chromomal abberation study will be conducted with GDMP.