Oxybis(methyl-2,1-ethanediyl) diacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria
- S. typhimurium TA 1535, TA 1537, TA1538, TA 98 and TA 100, with and without metabolic activation (Ames test): negative

- S. typhimurium TA1535, TA1537, TA98, TA100, and E.coli WP2 uvr A, with and without metabolic activation (Ames test): negative Read-across to tripropylene glycol diacrylate (Cas No. 42978-66-5)

Gene mutation in mammalian cells

- Chinese hamster lung fibroblasts (V79) cells, non-mutagenic, with and without metabolic activation, negative, HPRT assay, OECD TG 476

- Chinese hamster Ovary (CHO) cells, with and without metabolic activation (Sister Chromatide Exchange Assay): positive
- Mouse L5178Y cells, with and without metabolic activation (Mouse Lymphoma Assay): positive

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment.

Principles of method if other than guideline:

Dipropyleneglycol diacrylate was tested in vitro in the sister chromatid exchange assay using Chinese Hamster Ovary cells. The test article was tested at six dose levels with and without induced rat liver S-9 activation.

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

initial toxicity determination: without S-9 mix: 0.5, 1, 5, 10, 50, 100 nl/ml; with S-9 mix: 1, 5, 10, 50, 100, 500 nl/ml
SCE assay: without S-9 mix: 0.5, 1, 1.5, 2, 2.5, 3 nl/ml; with S-9 mix: 1, 10, 20, 30, 40, 50 nl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [ethanol]
- Justification for choice of solvent/vehicle: none given

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: triethylenemelamine (without S-9 mix), cyclophosphamide (with S-9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


all other information see: Any other information on materials and methods

Evaluation criteria:

A compound is assessed as positive if it meets the following criteria or requirements:
1. A minimum of two dose levels should show at least double the number of SEC's as is observed in the negative or solvent controls.
2. In the absence of the above, a positive dose response to a number of SCE's produced over a minimum of three of the doses. A student T-test is performed to compare the SCE frequency of test doses to the background frequencies and if P<0.05, the compound is assessed positive.

Statistics:

A total of 25 metaphases from each duplicate culture were read and the mean SCE/cell and standard deviation were calculated. The calculations were done on a Hewlett-Packard HP 75 programmable calculator. A pairwise T-test was performed to determine the P value.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with S-9 mix at 100 nl/ml; without S-9 mix: 5nl/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the initial cytotoxicity test, the test article showed a relative cell survival (RCS) of 22.57% at 100.0 nl/ml and 69.70% at 50.0 nl/ml in the activated
system and 27.95% at 5.0 nl/ml and 97.19 % at 1.0 nl/ml in the non-activated system. The results of the initial toxicity test were plotted on graph paper and a dose falling between 30 % and 50% reduction in the RCS was selected as the highest test dose for the SCE assay The sister chromatid exchange assay was, therefore, conducted at six decreasing dose levels starting at 50.0 nl/ml in the activated system and at 3 .0 nl/ml in the non-activated
system.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the initial cytotoxicity test, the test article showed a relative cell survival (RCS) of 22.57% at 100.0 nl/ml and 69.70% at 50.0 nl/ml in the activated
system and 27.95% at 5.0 nl/ml and 97.19 % at 1.0 nl/ml in the non-activated system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Assay results number of SCEs per cell at different concentrations tested

test material concentration [nl/ml]	Mean SCE per cell ± SD
50 nl/ml - 1 nl/ml; with metabolic activation
50	24.66 ± 4.60
40	18.28 ± 2.74
30	17.62 ± 3.37
20	15.84 ± 3.17
10	14.16 ± 2.57
1	16.22 ± 3.28
negative control	14.08 ± 3.16
positive control (CP 2.5 µg/ml)	47.06 ± 4.98
solvent control	12.44 ± 2.19
3 nl/ml - 0.5 nl/ml; without metabolic activation
3	17.98 ± 3.38
2.5	14.38 ± 2.55
2	12.24 ± 2.45
1.5	12.86 ± 2.26
1	11.30 ± 2.70
0.5	11.12 ± 2.50
negative control	10.66 ± 1.90
positive control (TEM 0.025 µg/ml)	52.94 ± 5.44
solvent control	10.12 ± 2.43

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: meets generally accepted scientific standards, well documented and acceptable for assessment

Principles of method if other than guideline:

The test substance was tested in the Salmonella/ mammalian-microsome mutagenicity assay using five tester strains, TA98, TA100, TA1535, TA1537 and TA1538, both with and without metabolic activation by Aroclor induced rat liver microsomes.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

60, 300, 1500, 3000 and 6000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [ethanol]
- Justification for choice of solvent/vehicle: none given

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9 mix for strains TA98, TA100, TA1535, TA1537 and TA1538

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S-9 mix for strains TA98 and TA1538

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 1,3-propane sultone

Remarks:

without S-9 mix for strains TA100 and TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S-9 mix for strain TA1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours


SELECTION AGENT (mutation assays): minimal medium

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

For a test article to be considered positive, it must cause at least a doubling in the average revertants per plate of at least one tester strain. This
increase in the average number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article,
and in those doses where the observed increase is less than three-fold, the response must be reproducible.

Statistics:

All platings were done in triplicate. For each triplicate plating, an average and standard deviation were calculated. The calculations were done on a
Hewlett-Packard HP-25 programmable calculator.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Max. revertants per plate in the different S. typhimurium strains tested

Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	vehicle control	59 ± 15	47 ± 9
	Test substance	78 ± 13 [60]	54 ± 9 [60]
	Positive Control	1290 ± 14 [0.04 µl; 1,3-Propane Sultone]	239 ± 12 [4 µg; 2-Aminoanthracene]
S. typhimurium TA100	vehicle control	209 ± 17	172 ± 19
	Test substance	237 ± 27 [300]	181 ± 11 [60]
	Positive Control	1405 ± 40 [0.04 µl; 1,3-Propane Sultone]	467 ± 32 [1 µg; 2-Aminoanthracene]
S. typhimurium TA98	vehicle control	19 ± 2	32 ± 3
	Test substance	18 ± 3 [60]	31 ± 3 [60]
	Positive Control	824 ± 44 [10 µg; 2-Nitrofluorene]	190 ± 30 [1 µg; 2-Aminoanthracene]
S. typhimurium TA1537	vehicle control	8 ± 3	13 ± 10
	Test substance	7 ± 2 [60]	6 ± 2 [60]
	Positive Control	297 ± 243 [75 µg; 9-Aminoacridine]	164 ± 25 [4 µg; 2-Aminoanthracene]
S. typhimurium TA1538	vehicle control	7 ± 3	15 ± 3
	Test substance	12 ± 4 [300]	16 ± 2 [300]
	Positive Control	1662 ± 76 [10 µg; 2-Nitrofluorene]	254 ± 9 [1 µg; 2-Aminoanthracene]

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

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: meets generally accepted scientific standards, well documented and acceptable for assessment

Principles of method if other than guideline:

Diproypleneglycol diacrylate was tested in the L5178Y TK+/- Mouse Lymphoma Mutagenesis assay with and without exogenous metabolic activation by Aroclor induced rat liver microsomes.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK) gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

initial toxicity determination: with and without S-9 mix: 0.001, 0.01, 0.1, 1.0, 10, 100 µl/ml
MLMA assay: without S-9 mix: 0.0013 - 0.01 µl/ml; with S-9 mix: 0.013 - 0.1 µl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [ethanol]
- Justification for choice of solvent/vehicle: none given

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): after 10-12 days


SELECTION AGENT (mutation assays): 5-trifluorothymidine


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Positive - if there is a positive dose response and one or more of the three highest doses exhibit a mutant frequency which is two-fold greater than the
background level.
Equivocal - if there is no dose response but any one or more doses exhibit a two-fold increase in mutant frequency over background.
Negative - if there is no dose response and none of the test cultures exhibit mutant frequencies which are two-fold greater than background.

Statistics:

All mutant frequency and toxicity data calculations were performed using a Texas Instruments TI-59 calculator.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to the read-across justification attached in chapter 13.2. of the IUCLID dossier

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 500 µg/plate

Vehicle controls validity:

not valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 500 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA1537, TA98, and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 500 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 500 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Please refer to the read-across justification attached in chapter 13.2. of the IUCLID dossier

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, Ta1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: see "Additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

GENETIC TOXICITY IN VIVO:
- Mouse, MNT in-vivo: negative (acc. OECD 474, BASF AG 2004); Read-across to tripropylene glycol diacrylate (Cas No. 42978-66-5)
- Mouse, MNT in-vivo: negative (acc. OECD 474, Covance 2007); Read-across to tripropylene glycol diacrylate (Cas No. 42978-66-5)
- Mouse, Single cell gel assay and micronucleus assay: negative (Tice 1997, Val. 2); Read-across to tripropylene glycol diacrylate (Cas No. 42978-66-5)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Physical state: liquid
- Analytical purity: >95%
- Lot/batch No.: 030061P040
- Stability under test conditions: the stability of the test substance throughout the study period and in the vehicle was verified analytically.
- Storage condition of test material: room temperature, protected from light

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 31 g (mean)
- Assigned to test groups randomly: yes, under following basis: randomization plan prepared with an appropriate computer program.
- Housing: Makrolon cages, type MI, housed individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, olive oil was selected as the vehicle, which had
been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 0.875 g/100 ml; 1.75 g/100 ml and 3.5 g/100 ml

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in olive oil:
- The low dose group was given 87.5 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of
0.875 g/100 ml.
- The intermediate dose group was given 175 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of
1.75 g/100 ml.
- The top dose groups were given 350 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 3.5 g/100 ml.

Duration of treatment / exposure:

one single administration

Frequency of treatment:

one single administration

Post exposure period:

24-48 hours

Dose / conc.:

87.5 mg/kg bw/day (nominal)

Dose / conc.:

175 mg/kg bw/day (nominal)

Dose / conc.:

350 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP) and vincristine (VCR) both dissolved in purified water were administered to male animals once intraperitoneally each in a
volume of 10 ml/kg body weight.
- Justification for choice of positive control(s): The stability of CPP and VCR is well-defined under the selected conditions, since both positive control
articles are well-established reference clastogens and aneugens respectively.
- Route of administration: intraperitoneal
- Doses / concentrations: CPP: 20 mg/kg bw for clastogenic effects; VCR: 0.15 mg/kg bw for aneugenic effects

Tissues and cell types examined:

In general, 2000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored .

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES:
The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of
350 mg/kg body weight and in the vehicle controls. In the test groups of 175 mg/kg and 87.5 mg/kg body weight and in the positive control groups,
the 24-hour sacrifice interval was investigated only.

DETAILS OF SLIDE PREPARATION:
The two femora were prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the
diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur). The suspension was mixed
thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µl fresh FCS. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution), rinsed in purified
water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5% Giemsa solution for 15 minutes.
After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2,000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an
index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression
may be identified and excluded from the evaluation.
- Number of small micronuclei (d=D/4) (d = diameter of micronucleus, D= cell diameter)
The size of micronuclei may indicate the possible mode of action of the test substance, i .e . a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis.
Since the absolute values shown have been rounded off but the calculations were made using the unedited values, deviations in the given relative
values can occur.

Evaluation criteria:

The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i .e. >=2000 polychromatic erythrocytes and a clear differentiation between polychromatic erythrocytes (PECs) and normochromatic erythrocytes (NECs).
-The ratio of PECs/NECs in the untreated animals (negative control) has to be within the normal range of the animal strain.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for
PECs and NECs.
- The two positive control substances have to induce a significant increase in the number of PECs containing small and large micronuclei within the
range of the historical control data or above.

A finding is considered positive if the following criteria are met:
- Significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.

A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN.
The asymptotic U test according to Mann-Whitney (modified rank test according to Wilcocon) was carried out to clarify the question whether there were significant differerences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes.
The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determinatian for the U test .

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

the test substance led to clinical signs

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

As a negative control, male mice were administered merely the vehicle, olive oil,by the same route, which

gave frequencies of micronucleated polychromatic erythrocytes within the historical control range.
Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity.

The administration of the test substance led to clinical signs, namely piloerection and squatting posture.

According to the results of the present study, the single intraperitoneal administration of Tripropylenglykoldiacrylat did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data.

A dose-dependent inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected from about of 87.5 mg/kg body weight onward.

Conclusions:

Thus, under the experimental conditions chosen in the study, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): TPGDA
- Analytical purity: approx. 87 %
- Lot/batch No.: P8960464SAP
- Expiration date of the lot/batch: 19 March 2008

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Frederick, USA
- Age at study initiation: Young adults, approx. 8 weeks old
- Weight at study initiation: males: 33.7 to 38.2 g; females: 23.1 to 28.1 g
- Assigned to test groups randomly: yes by using a computer program
- Housing: separated by gender, up to 5 animals per cage
- Diet: PMI Certified Rodent Diet (R) #5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17.7 - 26.1 °C
- Humidity: 30-70 %
- Air changes per hr: >/= 10
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil (CAS No. 8001-30-7); Supplier: Welch, Holme & Clarke; Lot No. 12-455; Storage at > 0 °C to 10 °C

Details on exposure:

Dose Preparation
Prior to dosing, the top stock of the test article, TPGDA, was prepared by adding the appropriate volume of the vehicle, corn oil, to a pre-weighed quantity of the test article and mixed, forming a solution. Lower concentrations were obtained by dilution with the vehicle. The formulations were held at room temperature prior to dosing.

Dose Analyses
The Sponsor was responsible for the determination and documentation of the identity, strength, purity, stability and uniformity of the test article and the determination of stability, homogeneity and concentration of the dosing preparations.

Duration of treatment / exposure:

one single administration

Frequency of treatment:

one single administration

Post exposure period:

24 h for all dose groups; additionally 48 h for an additional vehicle control and an additional 2000 mg/kg bw group

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Range-finder and main study

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Range-finder and main study

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Range-finder and main study

No. of animals per sex per dose:

Range-finding study: 3 males and 3 females per dose
Main study: 5 males per dose and harvest time point

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CAS No. 6055-19-2); Supplier: Sigma Aldrich; Lot No. 076K1050; Storage in a refrigerator set to maintain >0 to 10 °C;
the solvent was water

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Extraction of Bone Marrow
The hind limb bones (tibias) were removed for marrow extraction from five surviving animals in each treatment and control group. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal).

Preparation of Slides
Following centrifugation to pellet the marrow, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed in methanol, stained in May-Grünwald solution and Giemsa, and protected by mounting with coverslips. For control of bias, all slides were coded prior to analysis.

Slide Analysis
Slides prepared from the bone marrow collected from five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least 500 erythrocytes per animal.

Evaluation criteria:

Acceptable Controls
The vehicle control group mean must lie within the historical control range and will usually be less than 0.4% micronucleated PCEs. There must be a statistically significant elevation of the mean of the positive control group relative to the vehicle control group, and the positive control response must be consistent with historical positive control data.
Acceptable High Dose
Generally the high dose should reach the limit dose or produce some indication of toxicity, e.g., toxic signs and/or mortality in the test article dosed animals and/or a reduction in the PCE:NCE ratio. If there are solubility constraints, the highest dose tested will be the solubility limit or higher doses if a well-dispersed suspension is obtained that does not settle out rapidly.
Assay Evaluation Criteria
The criteria for a positive response is the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose related response. A test article that does not induce both of these responses is considered negative. Statistical significance is not the only determinant of a positive response; the Study Director also considers the biological relevance of the results in the final evaluation.

Statistics:

The following statistical methods were used to analyze the micronucleus data.
• Assay data analysis was performed using an analysis of variance (Winer, 1971) on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances.
• If the analysis of variance was statistically significant (p ≤ 0.05), Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time.
The 500, 1000, and 2000 mg/kg dose groups, as well as the positive control group, were compared with the vehicle control group at the 5% probability level.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose Range-finding Study:
Survival and Clinical Observations:
All animals appeared normal immediately after dosing and remained healthy until the end of the observation period.
Conclusion:
The high dose of the commercial product reached the maximum allowable dose of 2000 mg/kg bw, based on regulatory guidelines.


Micronucleus Assay:
- Survival and Clinical Observations:
All animals in all the dose groups appeared normal immediately after dosing and remained healthy until the appropriate harvest timepoint. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest timepoint.
- Results and Interpretation:
A commercial product of TPGDA did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (500, 1000, or 2000 mg/kg). TPGDA was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose of the test article analyzed.
The vehicle control group had approximately ≤0.09% micronucleated PCEs and the group mean was within the historical control range. The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard error of 1.93 ± 0.21%.

Table 1: Individual animal data after 24 and 48 h treatment

Treatment	Dose	Animal No.	# MN PCE/2000 PCE	Ratio PCE:NCE
vehicle control 24 h	corn oil 10 ml/kg bw	1	1	0.29
	corn oil 10 ml/kg bw	2	2	0.26
	corn oil 10 ml/kg bw	3	2	0.66
	corn oil 10 ml/kg bw	4	1	0.25
	corn oil 10 ml/kg bw	5	3	0.46
vehicle control 48 h	corn oil 10 ml/kg bw	1	3	0.56
	corn oil 10 ml/kg bw	2	1	0.57
	corn oil 10 ml/kg bw	3	3	0.31
	corn oil 10 ml/kg bw	4	1	0.26
	corn oil 10 ml/kg bw	5	1	0.50
positive control	cyclophosphamide 80 mg/kg bw	1	50	0.10
	cyclophosphamide 80 mg/kg bw	2	26	0.36
	cyclophosphamide 80 mg/kg bw	3	45	0.52
	cyclophosphamide 80 mg/kg bw	4	38	0.74
	cyclophosphamide 80 mg/kg bw	5	34	0.57
Test material 24 h	500 mg/kg bw	1	2	0.81
	500 mg/kg bw	2	0	0.44
	500 mg/kg bw	3	0	0.56
	500 mg/kg bw	4	0	0.38
	500 mg/kg bw	5	3	0.20
Test material 24 h	1000 mg/kg bw	1	1	0.30
	1000 mg/kg bw	2	0	0.49
	1000 mg/kg bw	3	0	0.76
	1000 mg/kg bw	4	1	0.42
	1000 mg/kg bw	5	1	0.64
Test material 24 h	2000 mg/kg bw	1	0	0.32
	2000 mg/kg bw	2	1	0.48
	2000 mg/kg bw	3	4	0.29
	2000 mg/kg bw	4	6	0.45
	2000 mg/kg bw	5	1	0.53
Test material 48 h	2000 mg/kg bw	1	0	0.53
	2000 mg/kg bw	2	1	0.36
	2000 mg/kg bw	3	1	0.45
	2000 mg/kg bw	4	2	0.31
	2000 mg/kg bw	5	1	0.26

PCE = Polychromatic erythrocyte

MN PCE = Micronucleated PCE

NCE = Normochromatic erythrocyte

Conclusions:

The test material was evaluated as negative in the mouse bone marrow micronucleus assay under the experimental conditions of this assay chosen.

Reference 3

Endpoint:

genetic toxicity in vivo, other

Remarks:

chromosome aberration and DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The test substance was applied dermally to Tg.AC mice (3 times a week for 20 weeks). Peripheral blood leukocytes were evaluated for DNA damage (single-strand breaks, alkali labile sites, DNA crosslinking) at weeks 4, 8, 12, 16, and 20 by using the alkaline (pH > 13) single cell gel (SCG) assay.
Peripheral blood polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were evaluated for the presence of micronuclei at week 20.

GLP compliance:

not specified

Type of assay:

other: Single cell gel assay and micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Tripropylene glycol diacrylate (TPGDA)
- Physical state: liquid
- Analytical purity: 80% pure monomer
- Impurities (identity and concentrations): hydroquinone (<200 ppm)
- Storage condition of test material: in the dark at 4-6 °C

Species:

mouse

Strain:

other: Tg.AC (v-Ha-ras)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic farms
- Age at study initiation: 12 weeks of age
- Assigned to test groups randomly: yes, under following basis: randomly assigned by body weight to a treatment or control group
- Housing: 3 to 5 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 4 °C (71 +-7 °F)
- Humidity (%): 50 +- 20 %
- Air changes (per hr): 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

dermal

Vehicle:

- Vehicle(s)/solvent(s) used: [acetone]
- Justification for choice of solvent/vehicle: the test substance is immiscible with water
- Concentration of test material in vehicle: 1, 5 and 10 μM in 200 μl vehicle (acetone)
- Amount of vehicle (if gavage or dermal): 200 μl vehicle (acetone)

Details on exposure:

TEST SITE
- Area of exposure: midscapular region to the base of the tail
- % coverage: 8 cm2
- Time intervals for shavings or clipplings: before the first treatment, no further data


REMOVAL OF TEST SUBSTANCE
no data


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1, 5 and 10 μM in 200 μl vehicle (acetone)
- Concentration (if solution): 1, 5 and 10 μM in 200 μl vehicle (acetone)
- Constant volume or concentration used: yes


USE OF RESTRAINERS FOR PREVENTING INGESTION: no data

Duration of treatment / exposure:

20 weeks

Frequency of treatment:

three (Monday, Wednesday, Friday) topical applications/week

Post exposure period:

no data

Dose / conc.:

1 other: µM

Remarks:

Nominal concentration

Dose / conc.:

5 other: µM

Remarks:

Nominal concentration

Dose / conc.:

10 other: µM

Remarks:

Nominal concentration

No. of animals per sex per dose:

3-8

Control animals:

yes, concurrent vehicle

Positive control(s):

12-O-tetra-decanoylphorbol-13-acetate
- Justification for choice of positive control(s): positive control for tumor induction in this transgenic mouse model
- Route of administration: dermal
- Doses / concentrations: 0.002 μM
This positive control substance is not a genotoxicant, but a tumor promotor.

Tissues and cell types examined:

At 4, 8, 12, 16, and 20 weeks of treatment (~ 3-4 hours after the treatment on Wednesday ; 27-28 hours after the treatment on Monday), 1-2 mm of
the terminal portion of the tail was snipped and blood collected for an evaluation of DNA damage in leukocytes or micronuclei in erythrocytes.

Details of tissue and slide preparation:

see any other information on materials and methods

Evaluation criteria:

Criteria for a positive response included a statistically significant trend test or Kruskall-Wallis test with at least one dose significantiy different from the concurcent control, or at least two doses significantly different from the concurcent control.

Statistics:

Significance was based on obtaining p< 0.05. The statistical analysis of micronuclei data was conducted by using a micronucleus assay data
management, and statistical analysis software system.
For single cell gel data, the statistical analysis was based on tail moment, a metric that takes into account both the amount of migrated DNA and
the length of DNA migration.

Sex:

female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

other: valid, but not useful as positive control for DNA damage

Additional information on results:

The positive control substance induced the expected increase in skin papilloma in the transgenic mouse model. Since it is known, that this substance does not cause DNA damage, but has only tumor promoting properties, it gave negative results in the micronucleus and single cell gel assay. Its usefulness as a positive control in this study is questionable.

The extent of DNA migration in leukocytes of mice treated by dermal application with TPGDA at 1, 5, or 10 μmol per mouse was not significantly different, either by trend test analysis or by a pairwise comparison of each treatment dose against the concurrent vehicle control, at any sample time.

TPA (0.002 μmol per mouse), the positive control for the tumorigenicity studies, also failed to significantly alter the extent of DNA migration or its intercellular dispersion in leukocytes of mice treated by dermal application.

After 20 weeks of treatment, the frequency of micronucleated PCE and NCE in blood were not increased in the

mice treated with TPGDA or TPA.

The percentage of PCE was increased in mice treated with TPGDA. This increase was highly significant. By a pairwise

comparison, the lowest effective dose of TPGDA inducing a significant increase in percentage of PCE was 10

μmol per mouse. TPA, at 0.002 μmol per mouse also induced a marginally nonsignificant increase in the percentage of PCE. This observed increase in the rate of erythropoiesis may reflect bone marrow/blood toxicity, a homeostatic mechanism in response to

the treatment-induced tumor burden, and/or a hematopoietic response to epidermal keratinocyte cytokines induced by tissue injury.

Conclusions:

In this study, the dermal application of the test substance, a multifunctional acrylate to female Tg.AC mice over a 20-week period, failed to induce a significant increase in DNA damage in circulating leukocytes at multiple sample times or chromosomal damage in proliferating
bone marrow cells. The absence of genotoxic damage in these two cell populations suggests that this acrylate is not genotoxic or that it is genotoxic but not readily absorbed across the skin and systemically distributed throughout the body.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Valid experimental data were available to assess the genetic toxicity in vitro:

 

- Gene mutation in bacteria:DPGDA was not mutagenic in an Ames test with and without metabolic activation, tested in concentrations of 60, 300, 1500, 3000 and 6000μg/plate in Salmonella typhimurium TA1535, TA 1537, TA 1538, TA 98 and TA 100; S9 fraction was from Aroclor induced rat liver microsomes (Microbiological Assoc. 1984, Val. 2). Cytotoxicity was not observed. The genotoxic potential in bacteria of the target substance DPGDA in vitro is supplemented with the available in vitro studies in bacteria for TPGDA. Based on data of the source substance, sufficient information is available to conclude DPGDA does not have any genotoxic potential.

 

- Gene mutation in mammalian cells:

An study was performed to investigate the potential of Oxybis(methyl-2,1-ethanediyl) diacrylate to induce gene mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in the established cell line V79, derived from Chinese hamster lung cells according to OECD TG 476 and in compliance with GLP.

The auxiliary metabolic system (S9-mix) used was derived from the liver of rats and treated with Phenobarbital/β-naphthoflavone. Mutant frequencies were assessed by cell growth in the presence of 6-thioguanine (6-TG). A pre-test was performed in order to determine the toxicity of the test item. In addition the pH-value and the osmolarity were measured.The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 15.6 μg/mL and 2000 μg/mL were tested concentration according to the OECD TG 476. In the main experiment, cells were treated with 0.8;1.6; 3.1; 6.3; 12.5; and 18.8 μg/mL without and 15.6; 31.3; 62.5; 125.0; 250.0 μg/mL with metabolic activation. The exposure duration was 4 hours (both with and without metabolic activation).

In the pre-test, no relevant toxic effect was noted up to the highest concentration in the presence and absence of metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The maximum test item concentration of the main experiment with metabolic activation was equal to 2000μg/mL. The concentration range of the main experiment without metabolic activation was limited by cytotoxicity.

No substantial and dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Oxybis(methyl-2,1-ethanediyl) diacrylate is considered to be non-mutagenic in this HPRT assay.

In another study, a sister chromatide exchange assay was conducted with Chinese Hamster Ovary cells, with and without metabolic activation (Microbiological Assoc. 1985, Val. 2). Dose levels of 50, 40, 30, 20, 10 and 1 nl/ml in the activated system and dose levels of 3.0, 2.5, 2.0 and 1.5 nl/ml in the nonactiveted system showed significant increases (p<0.05) in the frequencies of SCEs over that of their solvent controls. The positive control with and without activation produced a significant increase in the frequency of sister chromatid exchanges.

The test substance was also tested positive for genotoxicity in a mouse lymphoma assay using L5178Y cells at levels up to 0.01 µl/ml without metabolic activation and up to 0.1 µl/ml with metabolic activation (Microbiological Assoc. 1984, Val. 2). Cytotoxicity was observed.

 

In vivo:

There are no valid data available to assess the genetic toxicity of DPGDA in-vivo.

However, there are valid data available which assessed the genetic toxicity toxicity of the structurally related tripropylene glycol

diacrylate (TPGDA) (Cas No. 42978-66-5) in-vivo. These data were adopted from tripropylene glycol diacrylate

for DPGDA by read-across:

Tripropylene glycol diacrylate showed no mutagenic activity in in-vivo assays with rodents. In a mouse micronucleus assay on polychromatic erythrocytestripropylene glycol diacrylateled to a negative result after single oral administration of 87.5, 175, 350 mg/kg bw. Sampling times were 24 and 48 h (BASF AG 2004, Val. 1). As a negative control, male mice were administered merely the vehicle, olive oil,by the same route, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity. The administration of the test substance led to clinical signs. According to the results of the present study, the single intraperitoneal administration of tripropylene glycol diacrylate did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data. A dose-dependent inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected from about of 87.5 mg/kg body weight onward.

 

Another in vivo micronucleus test was also negative after oral administration of tripropylene glycol diacrylate. In this test doses of 2000, 1000 and 500 mg/kg bw were administered. Sampling time was 24 and 48 h after administration. 5 males per dose and harvest time point were used (Covance 2007, Val. 1). All animals in all the dose groups and controls appeared normal immediately after dosing and remained healthy until the appropriate harvest timepoint. TPGDA did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (500, 1000, or 2000 mg/kg). TPGDA was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose of the test article analyzed. The vehicle control group had approximately≤0.09% micronucleated PCEs and the group mean was within the historical control range. The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control.

In another study the test substance was applied dermally to Tg.AC mice (3 times a week for 20 weeks). Peripheral blood leukocytes were evaluated for DNA damage (single-strand breaks, alkali labile sites, DNA crosslinking) at weeks 4, 8, 12, 16, and 20 by using the alkaline (pH > 13) single cell gel (SCG) assay. Peripheral blood polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were evaluated for the presence of micronuclei at week 20 (Tice 1997, Val. 2). The extent of DNA migration in leukocytes of mice treated by dermal application with TPGDA at 1, 5, or 10 μmol per mouse was not significantly different, either by trend test analysis or by a pairwise comparison of each treatment dose against the concurrent vehicle control, at any sample time. TPA (0.002 μmol per mouse), the positive control for the tumorigenicity studies, also failed to significantly alter the extent of DNA migration or its intercellular dispersion in leukocytes of mice treated by dermal application.After 20 weeks of treatment, the frequency of micronucleated PCE and NCE in blood were not increased in the mice treated with TPGDA or TPA. The percentage of PCE was increased in mice treated with TPGDA. This increase was highly significant. By a pairwise comparison, the lowest effective dose of TPGDA inducing a significant increase in percentage of PCE was 10 μmol per mouse. TPA, at 0.002 μmol per mouse also induced a marginally nonsignificant increase in the percentage of PCE. This observed increase in the rate of erythropoiesis may reflect bone marrow/blood toxicity, a homeostatic mechanism in response to the treatment-induced tumor burden, and/or a hematopoietic response to epidermal keratinocyte cytokines induced by tissue injury.

 

Justification for classification or non-classification

The test substance was not genotoxic in in-vitro experiments using bacterial cells. In mammalian cells in-vitro however, genotoxic effects were seen in a Mouse Lymphoma Assay as well as in a Sister Chromatide Exchange assay.

The results of in vitro mouse lymphoma assay and Sister Chromatide Exchange assay, raising the concern of mutagenicity of DPGDA, can be clarified by results of in vivo testing. Though there are no data available to assess the genetoc toxicity of DPGDA in-vivo, there are data available which were adopted from the structurally similar TPGDA for DPGDA by read across: The behavior of the tested material TPGDA indicates without equivocation in the mouse micronucleus tests and in the Single cell gel assay that this chemical is nongenotoxic in the whole animal.

As a conclusion, DPGDA does not fulfill the requirements to be classified regarding this endpoint.