Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In three recent in vitro GLP studies conducted according to OECD guidelines, TMPTMA was found negative in bacterial reverse mutation test and mammalian gene mutation assay (HPRT) and was found positive in chromosome aberration test on human lymphocytes only in presence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 20 to June 25, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9), obtained from Molecular Toxicology Incorporated, USA
Test concentrations with justification for top dose:
- Range-finder experiment and experiment 1: 1.6, 8, 40, 200, 1000 and 5000 µg/plate (with and without S-9)
- Experiment 2: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate (with and without S-9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Solubility of propylidynetrimethyl trimethacrylate in DMSO = 100 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate incorporation method; as the results of Experiment 1 were negative, treatments in the presence of S-9 in experiment 2 included a pre-incubation step (incubation for 1 hour at 37±1°C).

DURATION
For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate. Incubation was carried out with shaking in an anhydric incubator. All treatments were completed within 6 hours of the end of the incubation period.
After plating with test substance or control, the plates were inverted and incubated at 37±1°C in the dark for 3 days.

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS:
Range-finding test: triplicate plates; negative (vehicle) and positive controls were included in quintuplicate and triplicate, respectively.
Main experiments: triplicate plates; negative (vehicle) controls were included in quintuplicate, and positive controls were included in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants; thinning of background bacterial lawn.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
- Dunnett's test gave a significant response (p=< 0.01) which was concentration related
- The positive trend/effects described above were reproducible.
Negative: If all of the above criteria were not met
Statistics:
Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels.
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:
cytotoxicity
Remarks:
Marked reduction in revertant numbers was observed at 2500 µg/plate and above in strains TA1535 and TA1537 in the presence of S-9 and at 5000 µg/plate in strain TA1537 in the absence of S-9 in experiment 2
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Precipitation of the test article was observed on all test plates treated at 5000 µg/plate in the Range-Finder Experiment and Experiment 1 and at 2500 µg/plate and above in Experiment 2.
In the range-finder experiment and in experiment 1, no evidence of toxicity was observed as would normally manifest as a diminution of the background bacterial lawn or a marked reduction in revertant numbers.
In the second experiment, evidence of toxicity in the form of a marked reduction in revertant numbers was observed at 2500 µg/plate and above in strains TA1535 and TA1537 in the presence of S-9 and at 5000 µg/plate in strain TA1537 in the absence of S-9.
Conclusions:
Under the test conditions, propylidynetrimethyl trimethacrylate is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation(EC) N° (1272-2008). 
Executive summary:

In a GLP study performed according to OECD guideline 471, propylidynetrimethyl trimethacrylate was tested for mutagenicity using Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with the plate incorporation and preincubation methods in the presence and absence of metabolic activation system (S-9 mix). Due to absence of toxicity in the range-finder test, using TA100 tested at concentrations of 1.6, 8, 40, 200, 1000 and 5000 µg/plate with and without S-9, all strains in experiment 1 were tested with and without S-9 at these concentrations. In order to examine more closely those concentrations of propylidynetrimethyl trimethacrylate approaching the maximum test concentration, the following concentrations were tested in the second experiment (using the preincubation method (1h at 37°C) when S-9 was used): 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate, with and without S-9. Precipitation of the test article was observed on all test plates treated at 5000 µg/plate in the Range-Finder Experiment and Experiment 1 and at 2500 µg/plate and above in Experiment 2. In the range-finder experiment and in experiment 1, no evidence of toxicity was observed as would normally manifest as a diminution of the background bacterial lawn or a marked reduction in revertant numbers. In the second experiment, evidence of toxicity in the form of a marked reduction in revertant numbers was observed at 2500 µg/plate and above in strains TA1535 and TA1537 in the presence of S-9 and at 5000 µg/plate in strain TA1537 in the absence of S-9. The positive controls induced the appropriate responses in the corresponding strains. Propylidynetrimethyl trimethacrylate showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S-9. Under the test conditions, propylidynetrimethyl trimethacrylate is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation(EC) N° (1272-2008). 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 June 2010 - 6 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fractions of rat liver microsomes induced by Aroclor 1254 (Molecular Toxicology Incorporated, USA)
Test concentrations with justification for top dose:
Range-finding test: from 2.902 to 800.0 µg/mL
Main experiment: without S9: 40, 50, 60, 70, 80, 90, 100, 140, 180 and 250 µg/mL; with S9: 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 650 and 800 µg/mL.
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Untreated negative controls:
other: not applicable
Negative solvent / vehicle controls:
yes
True negative controls:
other: not applicable
Positive controls:
yes
Positive control substance:
other: - without S9 mix: 4-Nitroquinoline 1-oxide (NQO), 2.5 and 5 µg/mL, - with S9 mix: cyclophosphamide at 10, 20 and 30 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum and 50 µg/mL gentamycin)

DURATION
- Exposure duration: 3 h with and without S9; 20 h without S9
- Expression time (cells in growth medium): 17 h after the 3-h treatment;
- Fixation time (start of exposure up to harvest of cells): 20 h

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1.5 normal cell cycles

NUMBER OF CELLS EVALUATED: analysis of 200 metaphases/dose-level, with 100 metaphases/culture whenever possible.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- metaphase gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverisations)
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:
For valid data, the test article was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range was observed in both replicate cultures
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p = 0.05)
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
The test article was considered as positive in this assay if all of the above criteria were met. The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis.
Statistics:
The statistical significance of increases in the percentage of cells with structural aberrations for any data set was only taken into consideration if the frequency of aberrant cells in both replicate cultures at one or more concentrations exceeds the normal range. The statistical method used was Fisher's exact test.
Species / strain:
lymphocytes: Human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: Human
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
No marked changes in osmolality or pH were observed at the highest concentration tested (800.0 µg/mL), compared to the concurrent vehicle controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY AND PRECIPITATION: see tables 1 and 2

Table 1: Mitotic Index Determinations - Range-Finder Experiment

Treatment

Mitotic index (%)

(µg/mL)

3+17 hours, -S-9

3+17 hours, +S-9

20+0 hours, -S-9

 

A

B

MIH*

A

B

MIH*

A

B

MIH*

Vehicle

7.7

4.8

-

5.9

3.9

-

1.9

3.8

-

2.902

4.4

NT

30

NS

NT

-

NS

NT

-

4.837

5.6

NT

10

NS

NT

-

NS

NT

-

8.062

5.9

NT

6

NS

NT

-

4.1

NT

0

13.44

6.0

NT

4

NS

NT

-

3.2

NT

0

22.39

5.4

NT

14

NS

NT

-

5.1

NT

0

37.32

4.3

NT

31

NS

NT

-

2.8

NT

2

62.21

3.3

NT

47

5.8

NT

0

2.3

NT

19

103.7

2.3

NT

63

5.9

NT

0

2.5

NT

12

172.8

3.2

NT

49 P

6.3

NT

0 P

2.7

NT

5 P

288.0

1.3

NT

79 P

4.2

NT

14 P

1.6

NT

44 P

480.0

0.1

NT

98 PEH

4.1

NT

16 PE

1.5

NT

47 PH

800.0

0.2

NT

97 PEH

1.5

NT

69 PE

1.5

NT

47 PH

NT = Not tested

NS = Not scored

P = Precipitation observed at treatment

E = Precipitation observed at the end of treatment incubation

H = Precipitation observed at harvest

*Mitotic inhibition (%) = [1 - (mean MI T/mean MI C)] x 100% (where T = treatment and C = negative control)

Table 2: Mitotic Index Determinations - Main experiment

Treatment

(µg/mL)

Mitotic index (%)

3+17 hours, -S-9

3+17 hours, +S-9

A

B

MIH*

A

B

MIH*

Vehicle

9.8/6.1

9.2/11.3

-

10.2/9.0

12.1/10.0

-

10.00

NS

NS

-

NT

NT

-

20.00

NS

NS

-

NT

NT

-

40.00

9.7

10.2

0

NT

NT

-

50.00

10.2

10.7

0

11.2

10.5

0

60.00

9.9

9.0

0 P

NT

NT

-

70.00

7.7

7.7

15 P

NT

NT

-

80.00

7.8

8.5

10 P#

NT

NT

-

90.00

7.9

5.7

25 P#

NT

NT

-

100.0

4.3

2.9

60 P#

11.9

11.4

0 P

140.0

2.6

2.9

70 P

NT

NT

-

150.0

NT

NT

-

11.1

12.1

0 P

180.0

2.7

2.4

72 P

NT

NT

-

200.0

NT

NT

-

10.4

9.7

3 P

250.0

2.3

1.7

78 P

9.4

9.4

9 P

300.0

NT

NT

-

10.4

8.5

8 P

350.0

NT

NT

-

9.9

10.3

2 P#

400.0

NT

NT

-

10.6

7.2

14 P

450.0

NT

NT

-

9.2

7.5

19 P

500.0

NT

NT

-

10.0

9.9

4 P

650.0

NT

NT

-

6.8

6.3

37 P#

800.0

NT

NT

-

6.0

4.3

50 P#

NS = Not scored

P = Precipitation observed at treatment

*Mitotic inhibition (%) = [1 - (mean MI T/mean MI C)] x 100% (where T = treatment and C = negative control)

# Highlighted concentrations selected for analysis

Table 3: main experiment results without S-9

Treatment

(µg/mL)

Replicate

Cells

Scored

Cells with

Aberrations

Including

Gaps

Cells with

Aberrations

Excluding

Gaps

MIH

(%)

Vehicle

A

100

1

1

 

B

100

2

1

Totals

200

3

2

-

80.00

A

100

3

3

 

B

100

4

4 #

Totals

200

7

7

10

90.00

A

100

2

2

 

B

100

0

0

Totals

200

2

2

25

100.0

A

100

1

1

 

B

100

3

3

Totals

200

4

4

60

NQO, 2.50

A

48

20 #

19 #

 

B

49

19 #

19 #

Totals

97

39

38a

 

Binomial Dispersion Test ¿² = 3.19, Not significant

a Statistical significance p = 0.001

# Numbers highlighted exceed historical negative control range

Table 4: main experiment results with S-9

Treatment

(µg/mL)

Replicate

Cells

Scored

Cells with

Aberrations

Including

Gaps

Cells with

Aberrations

Excluding

Gaps

Significance

§

MIH

(%)

Vehicle

A

100

1

1

 

 

B

100

0

0

Totals

200

1

1

 

-

350.0

A

100

6 #

4 #

 

 

B

100

1

1

Totals

200

7

5

NS

2

650.0

A

94

18 #

15 #

 

 

B

100

14 #

13 #

Totals

194

32

28

p = 0.001

37

800.0

A

49

20 #

20 #

 

 

B

44

21 #

20 #

Totals

93

4

4

p = 0.001

50

CPA, 20.0

A

43

21 #

20 #

 

 

B

29

21 #

20 #

Totals

72

42

40

p = 0.001

Binomial Dispersion Test ¿² = 3.40, NS

§ Statistical significance

NS = Not significant

# Numbers highlighted exceed historical negative control range

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
positive with metabolic activation

Under these experimental conditions, propylidynetrimethyl trimethacrylate did not induce chromosome aberrations in cultured human lymphocytes without metabolic activation but induced chromosome aberrations in cultured human lymphocytes with metabolic activation.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, human primary lymphocytes were exposed to propylidynetrimethyl trimethacrylate dissolved in DMSO.

In a range-finding experiment, human lymphocyte cultures prepared from the pooled blood of three female donors were exposed to the test item for 3 h, with or without S9, or for 20 h without S9. Cells exposed for 3 h were harvested 20 h after the beginning of the treatment. Twelve concentrations were tested in the range of 2.902 - 800.0 µg/mL.

In the main experiment, cells were exposed for 3 h with or without S9, then rinsed and harvested 20 h after the beginning of the treatment.Concentrations from 40 to 250 µg/mL were tested without S9 and concnetrations from 50 to 800 µg/mL were tested with S9.

Positive controls (4-Nitroquinoline 1-oxide without S9 and cyclophosphamide with S9) induced the appropriate response.

Treatment of cells with propylidynetrimethyl trimethacrylate without S9 in the main experiment resulted in frequencies of cells with structural aberrations that were generally similar to those observed in concurrent vehicle control cultures for all concentrations analysed. Numbers of aberrant cells (excluding gaps) in treated cultures fell within the normal range with the exception of one culture at the lowest concentration analysed (80.00 µg/mL). However, the aberration frequency (excluding gaps) in the replicate culture at this concentration and at the two higher concentrations fell within the normal range; therefore this isolated observation was not considered biologically relevant.

Treatment in the presence of S-9 resulted in frequencies of cells with structural aberrations that were significantly higher (p = 0.001) than those observed in concurrent vehicle control cultures at the highest two concentrations analysed (650.0 and 800.0 µg/mL). Numbers of aberrant cells (excluding gaps) exceeded the normal range in one culture at the lowest concentration analysed (350.0 µg/mL) and in both cultures at 650.0 and 800.0 µg/mL and there was clear evidence of a concentration-related response.

No increases in the frequency of cells with numerical aberrations were observed with and without S9.

Under the test conditions, propylidynetrimethyl trimethacrylate was not clastogenic in human lymphocytes exposed in vitro without metabolic activation but was clastogenic in presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 June 2010 to August 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co. Cells
- Type and identity of media: RPMI 1640 medium
RPMI A: Penicillin (100 units/mL), streptomycin (100 µg/mL), amphotericin B (2.5 µg/mL) and pluronic acid (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10% v/v), penicillin (100 units/mL), streptomycin (100 µg/mL), amphotericin B (2.5 µg/mL) and pluronic acid (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20% v/v), penicillin (100 units/mL), streptomycin (100 µg/mL) and amphotericin B (2.5 µg/mL)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Yes; each batch was purged of TK- mutants
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
2% S9 fraction of Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Range-finder experiment: 0, 105.6, 211.3, 422.5 and 845 µg/mL (with and without S-9)
Main study:
- Experiment 1: without S-9: 0, 30, 60, 90, 120 and 150 µg/mL; with S-9: 0, 100, 200, 250, 300, 350, 400 and 450 µg/mL
- Experiment 2: without S-9: 0, 30, 50, 70, 90 and 110 µg/mL; with S-9: 0, 100, 150, 200, 300, 350, 400, 450 and 500 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: test material was poorly soluble in aqueous solutions
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours, 37 ± 1 ºC
- Expression time (cells in growth medium): 7 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
- Selection time (if incubation with a selection agent): 12-13 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Duplicates (single cultures only used for positive control treatments)

NUMBER OF CELLS EVALUATED: 20000 cells/well plated for survival, viability and 6TG resistance

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency

OTHER: Cell viability were identified by eye using background illumination and counted; cell densities were determined using a coulter counter.
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p = 0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p = 0.05)
3. The effects described above were reproducible.

- Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
The experimental data was analysed using UKEMS recommended statistical guidelines.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No data
- Water solubility: No data;
- Precipitation: Yes; in the range-finding study, precipitation, at time of treatment, was observed at concentrations from 110 and 100 µg/mL in the absence and presence of S-9, respectively.

RANGE-FINDING/SCREENING STUDIES: Five concentrations were tested with and without S-9 ranging from 0 to 845 µg/mL (limited by solubility in culture medium).
- precipitation, at time of treatment and after incubation period, was observed at concentrations from 211.3 to 845 µg/mL and at 845 µg/mL in the absence and presence of S-9, respectively.
- Without S-9: 21% relative survival (RS) was found at 211.3 µg/mL; with S-9: highest concentration provided 6% RS
- See table 1 for more details

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: without S-9, 15 % RS was obtained at 150 µg/mL in the first experiment whereas 11 % RS was obtained at 110 µg/mL in the second experiment. with S-9, 16 % RS was obtained at 450 µg/mL in the first experiment and 10 % RS was obtained at 500 µg/mL in the second experiment.

Table 1: RS values - range-finder experiment

Treatment

(µg/mL)

-S-9

% RS

+S-9

% RS

0

100

100

105.6

47

86

211.3 P, PP

21 (P,PP)

62 (P)

422.5 P

NE (P,PP)

19 (P)

845 P, PP

NE (P,PP)

6 (P,PP)

% RS: Percentage Relative Survival

P: Precipitation observed at time of treatment

PP: Precipitation observed following treatment incubation period

NE: Not evaluated for mutant frequency due to excessive toxicity. Presented for information only

Table 2: Summary of mutation data

Experiment 1: (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RS

MF§

 

%RS

MF§

0

 

100

0.21

 

0

 

100

2.25

 

30

 

83

0.58

NS

100

P

76

1.58

NS

60

 

56

1.39

NS

200

P

50

2.69

NS

90

 

35

0.95

NS

250

P

39

0.69

NS

120

P

23

1.41

NS

300

P

28

1.45

NS

150

P

15

0.62

NS

350

P

27

2.20

NS

 

 

 

 

 

400

P

25

1.57

NS

 

 

 

 

 

450

P

16

1.53

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

91

10.42

 

2

 

65

15.61

 

0.15

 

66

12.59

 

3

 

48

21.91

 

Experiment 2: (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RS

MF§

 

%RS

MF§

0

 

100

0.94

 

0

 

100

1.30

 

30

 

87

1.61

NS

100

P

69

0.93

NS

50

 

51

0.14

NS

150

P

60

2.50

NS

70

 

35

0.91

NS

200

P

56

1.76

NS

90

 

18

2.72

NS

300

P

32

0.80

NS

110

P

11

2.10

NS

350

P

30

0.64

NS

 

 

 

 

 

400

P

17

0.68

NS

 

 

 

 

 

450

P

18

1.85

NS

 

 

 

 

 

500

P

10

5.20

*

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

38

28.27

 

2

 

96

20.03

 

0.15

 

69

12.60

 

3

 

79

38.96

 

§6-TG resistant mutants/106viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

P: Precipitation observed at time of treatment

NS: Not significant

*: Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level

Conclusions:
Under the test conditions, propylidynetrimethyl trimethacrylate is not considered as mutagenic in L5178Y cells according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).
Executive summary:

In an in vitro mammalian cell gene mutation test, HPRT, performed according to OECD guideline 476, in compliance with GLP, mouse lymphoma L5178Y cells were exposed to propylidynetrimethyl trimethacrylate in DMSO at concentrations of 0, 105.6, 211.3, 422.5 and 845 µg/mL in RPMI 1640 medium with and without 2% S-9 metabolic activation for a preliminary cytotoxicity test. In the main test, two experiments were performed at the following concentrations:

- Experiment 1 (3-h exposure): without S-9: 0, 30, 60, 90, 120 and 150 µg/mL; with S-9: 0, 100, 200, 250, 300, 350, 400 and 450 µg/mL

- Experiment 2 (3-h exposure): without S-9: 0, 30, 50, 70, 90 and 110 µg/mL; with S-9: 0, 100, 150, 200, 300, 350, 400, 450 and 500 µg/mL

Positive controls used to validate the experiments were benzo(a)pyrene at 2 and 3 µg/mL without metbolic activation and 4-nitroquinoline-N-oxide at 0.1 and 0.15 µg/mL with metabolic activation.

When tested up to toxic concentrations, no significant increases in mutant frequency were observed at any concentration analysed in the absence and presence of S-9 in Experiment 1 and in the presence of S-9 in Experiment 2 and there were no significant linear trends. In the absence of S-9 in Experiment 2, a small but statistically significant increase in mutant frequency was observed at the highest concentration analysed (500 µg/mL, giving 10% RS) but there was no significant linear trend. As mentioned previously, lower concentrations of 400 and 450 µg/mL gave 17% and 18% RS, respectively, but no significant increases in mutant frequency were observed at these concentrations. Furthermore, the mutant frequency value at 500 µg/mL (5.20) only marginally exceeded the historical control mean value and fell within the historical negative control ranges. Overall, the data did not fulfil all of the criteria for a positive result and the small increase in mutant frequency in the absence of S-9 in Experiment 2 was not reproduced between experiments, therefore this isolated observation was not considered biologically relevant.

Under the test conditions, propylidynetrimethyl trimethacrylate is not considered as mutagenic in L5178Y cells according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Two in vivo studies (in vivo micronucleus assay and in vivo/in vitro UDS assay in rat liver) showed negative results.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 01 to March 22, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vivo micronucleus study
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HARLAN WINKELMAN, Borchen, Germany
- Age at study initiation: 23-33 g
- Assigned to test groups randomly: Yes
- Housing: Housed in a group of 5 mice/sex in Macaroon Type III cage
- Diet (e.g. ad libitum): Pelleted standard diet (ALTROMIN, Lage/Lippe, Germany)
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 55 ± 10
- Photoperiod (hours dark / hours light): 12 hours dark/12 hours light
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: Nontoxic to animals
- Source: SIGMA, Germany
- Amount of vehicle (if gavage or dermal): 33.3 mL/kg bw
- Lot/batch no. (if required): 36H0738
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material was prepared and diluted with carboxymethylcellulose.
Duration of treatment / exposure:
24 hours (in all test groups) and 48 hours (in 2000 mg/kg bw group)
Frequency of treatment:
Once
Post exposure period:
Not applicable
Remarks:
Doses / Concentrations:
0, 200, 600 and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA (cyclophosphamide)
- Source: SIGMA, Germany
- Analytical purity: At least 98%
- Route of administration: i.p.
- Doses / concentrations: 30 mg/kg bw
- Volume administered: 10 mL/kg bw
- Lot/batch no.: 087H0207
Tissues and cell types examined:
- Femora bones were removed for marrow extraction and the prepared slides were examined for polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs) and total erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose selection was based on a preliminary range-finding test conducted on 3 mice/sex/dose at 2000 mg/kg bw. No toxic signs were recorded.

TREATMENT AND SAMPLING TIMES: Femora were removed for marrow extraction from single animals in each treatment and control group at 24 or 48 hours after exposure.

DETAILS OF SLIDE PREPARATION: Bone marrow cells extracted, preparations smeared on slides and air-dried. The slides were stained with May-Grunwald solution/ Giemsa and coded.

METHOD OF ANALYSIS: Slides were scanned using Olympus microscopes to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) per animal. In addition, PCE:NCE ratio was determined in the same sample and expressed as NCE/1000 PCEs.
Evaluation criteria:
- Criteria for a positive response: Detection of either a statistically significant dose related increase in the number of micronucleated PCEs or a reproducible statistically significant positive response for at least one of the test points.
- Test article that does not induce both of these responses is considered negative.
- Both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the 5% level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No

RESULTS OF DEFINITIVE STUDY
- Frequency of micronuclei in polychromatic erythrocytes and PCE:NCE ratio: See table 1

Table 1: Micronucleus Assay - summary table

 

Treatment 

Dose

Harvest time (hours)

% Micronucleated PCEs (mean of 2000 PCEs per animal)

Ratio PCE:NCE (mean)

males

females

males

females

all

small

all

small

Vehicle control

Carboxymethylcellulose 33.3 mL/kg bw

 24 

0.16

0.14

0.16

0.14

1.14

1.35

Positive control

Cyclophosphamide 30 mg/kg bw

 24

1.01

0.94

0.98

0.92

1.35

1.45

Test Article 

200 mg/kg bw

 24

0.18

0.13

0.14

0.11

1.19

0.99

600 mg/kg bw

 24

0.09

0.07

0.12

0.09

0.86

0.93

2000 mg/kg bw

 24

0.12

0.11

0.1

0.09

0.89

1.11

 48

0.16

0.16

0.12

0.12

0.58

1.19

Conclusions:
Under the test conditions, trimethylolpropantrimethacrylate is not considered as mutagenic in the mouse bone marrow micronucleus test.
Executive summary:

In a bone marrow micronucleus test, performed according to OECD guideline 474 and in compliance with GLP, groups of NMRI mice (5/sex/dose) were given a single oral (gavage) dose of trimethylolpropantrimethacrylate in carboxymethylcellulose at doses of 0, 200, 600 and 2000 mg/kg bw. Bone marrow was extracted after 24 hours (in all test groups) or 48 hours (in 2000 mg/kg bw group) of exposure and the prepared slides were scanned to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) for each animal. In addition, PCE:NCE ratio was determined in the same sample and expressed as NCE/1000 PCEs. A preliminary range-finding test was also conducted on 3 mice/sex/dose at 2000 mg/kg bw in which no toxic signs were recorded.

 

No statistically significant increases in the frequency of micronucleated PCEs were observed at any dose levels. PCE:NCE ratio was slightly affected at a dose level of 2000 mg/kg bw at 24 and 48 hours indicating slight cytotoxicity of the test material. Positive control (cyclophosphamide, 30 mg/kg bw) induced the appropriate response.

 

Under the test conditions, trimethylolpropantrimethacrylate is not considered as mutagenic in the mouse bone marrow micronucleus test according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames test (Bowen 2010) :

In a GLP study performed according to OECD guideline 471, propylidynetrimethyl trimethacrylate was tested for mutagenicity using Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with the plate incorporation and preincubation methods in the presence and absence of metabolic activation system (rat S-9 mix). Due to absence of toxicity in the range-finder test, using TA100 tested at concentrations of 1.6, 8, 40, 200, 1000 and 5000 µg/plate with and without S-9, all strains in experiment 1 were tested with and without S-9 at these concentrations. In order to examine more closely those concentrations of propylidynetrimethyl trimethacrylate approaching the maximum test concentration, the following concentrations were tested in the second experiment (using the preincubation method (1h at 37°C) when S-9 was used): 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate, with and without S-9. Precipitation of the test article was observed on all test plates treated at 5000 µg/plate in the Range-Finder Experiment and Experiment 1 and at 2500 µg/plate and above in Experiment 2. In the range-finder experiment and in experiment 1, no evidence of toxicity was observed as would normally manifest as a diminution of the background bacterial lawn or a marked reduction in revertant numbers. In the second experiment, evidence of toxicity in the form of a marked reduction in revertant numbers was observed at 2500 µg/plate and above in strains TA1535 and TA1537 in the presence of S-9 and at 5000 µg/plate in strain TA1537 in the absence of S-9. The positive controls induced the appropriate responses in the corresponding strains. Propylidynetrimethyl trimethacrylate showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S-9.

In vitro gene mutation test (HPRT, Lloyd 2010a):

In the HPRT performed according to OECD guideline 476 and in compliance with GLP, mouse lymphoma L5178Y cells were exposed to propylidynetrimethyl trimethacrylate in DMSO. When tested up to toxic concentrations, no significant increases in mutant frequency were observed at any concentration analysed in the absence and presence of S-9 in Experiment 1 and in the presence of S-9 in Experiment 2 and there were no significant linear trends. In the absence of S-9 in Experiment 2, a small but statistically significant increase in mutant frequency was observed at the highest concentration analysed (500 µg/mL, giving 10% RS) but there was no significant linear trend. As mentioned previously, lower concentrations of 400 and 450 µg/mL gave 17% and 18% RS, respectively, but no significant increases in mutant frequency were observed at these concentrations. Furthermore, the mutant frequency value at 500 µg/mL (5.20) only marginally exceeded the historical control mean value and fell within the historical negative control ranges. Overall, the data did not fulfil all of the criteria for a positive result and the small increase in mutant frequency in the absence of S-9 in Experiment 2 was not reproduced between experiments, therefore this isolated observation was not considered biologically relevant. Under the test conditions, propylidynetrimethyl trimethacrylate is not considered as mutagenic in L5178Y cells.

In vitro gene mutation study in mammalian cells (MLA/TK, Myhr 1979):

An old MLA/TK study was performed on TMPTMA. In this study, the test compound induced an increase in mutations at the TK locus in L5178Y mouse lymphoma cells at doses of 100 to 200 nl/ml with microsomal activation. This dose range was moderately-to-highly toxic to the cells.

This results is supposed as a false positive results, known with acrylates (Johannsen FR, Vogt B, Waite M and Deskin R (2008). Mutagenicity assessment of acrylate and methacrylate compounds

and implications for regulatory toxicology programs. Reg. Toxicol. Pharmacol. 50:322-3").

In vitro chromosomal aberrations test (Lloyd 2010b)

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, human primary lymphocytes were exposed to propylidynetrimethyl trimethacrylate dissolved in DMSO. Treatment of cells with propylidynetrimethyl trimethacrylate without S9 in the main experiment resulted in frequencies of cells with structural aberrations that were generally similar to those observed in concurrent vehicle control cultures for all concentrations analysed. Numbers of aberrant cells (excluding gaps) in treated cultures fell within the normal range with the exception of one culture at the lowest concentration analysed (80.00 µg/mL). However, the aberration frequency (excluding gaps) in the replicate culture at this concentration and at the two higher concentrations fell within the normal range; therefore this isolated observation was not considered biologically relevant. Treatment in the presence of S-9 resulted in frequencies of cells with structural aberrations that were significantly higher (p = 0.001) than those observed in concurrent vehicle control cultures at the highest two concentrations analysed (650.0 and 800.0 µg/mL). Numbers of aberrant cells (excluding gaps) exceeded the normal range in one culture at the lowest concentration analysed (350.0 µg/mL) and in both cultures at 650.0 and 800.0 µg/mL and there was clear evidence of a concentration-related response. No increases in the frequency of cells with numerical aberrations were observed with and without S9.

Under the test conditions, propylidynetrimethyl trimethacrylate was not clastogenic in human lymphocytes exposed in vitro without metabolic activation but was clastogenic in presence of metabolic activation.

In vivo micronucleus test (Hamann 2000):

In a bone marrow micronucleus test, performed according to OECD guideline 474 and in compliance with GLP, groups of NMRI mice (5/sex/dose) were given a single oral (gavage) dose of trimethylolpropantrimethacrylate in carboxymethylcellulose at doses of 0, 200, 600 and 2000 mg/kg bw. Bone marrow was extracted after 24 hours (in all test groups) or 48 hours (in 2000 mg/kg bw group) of exposure and the prepared slides were scanned to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) for each animal. In addition, PCE:NCE ratio was determined in the same sample and expressed as NCE/1000 PCEs. A preliminary range-finding test was also conducted on 3 mice/sex/dose at 2000 mg/kg bw in which no toxic signs were recorded.

No statistically significant increases in the frequency of micronucleated PCEs were observed at any dose levels. PCE:NCE ratio was slightly affected at a dose level of 2000 mg/kg bw at 24 and 48 hours indicating slight cytotoxicity of the test material. Positive control (cyclophosphamide, 30 mg/kg bw) induced the appropriate response.

Under the test conditions, trimethylolpropantrimethacrylate is not considered as mutagenic in the mouse bone marrow micronucleus test.

In vivo UDS test (Volkner 2000):

In an Unscheduled DNA Synthesis test, performed according to OECD Guideline 486 and in compliance with GLP, groups of Wistar HanIbm: WIST (SPF) male rats (4/dose) were given a single oral (gavage) dose of trimethylolpropantrimethacrylate in polyethylene glycol 400 at concentrations of 0, 500 and 2000 mg/kg bw. After a treatment period of 2 and 16 hours, primary hepatocytes cultures from three animals/dose were established in Williams medium E (WME) and exposed for 4 hours to 3HTdR (methyl-3H-thymidine). Stained triplicate cultures were examined microscopically under oil immersion and UDS was measured by automatic counter and expressed as net nuclear grain count. A preliminary range-finding test was also conducted on 2 rats at 2000 mg/kg bw. Reduction of spontaneous activity was recorded after 1 and 24 hours of exposure.

Positive controls (N,N'-Dimethylhydrazinedihydrochloride, 40 mg/kg bw and 2-acetylaminofluorene, 100 mg/kg bw) induced the appropriate responses. No dose level of the test item revealed UDS induction in the hepatocytes of the treated animals as compared to the concurrent vehicle controls.

Under the test conditions, trimethylolpropantrimethacrylate is not considered as mutagenic in UDS test.

Justification for classification or non-classification

As the positive result found in vitro in a chromosome aberration test on human lymphocytes in presence of metabolic activation was not confirmed in two in vivo tests (micronucleus assay and UDS assay ), TMPTMA is not classified according to the CLP Regulation (EC) N° 1272-2008.