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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
06 October to 18 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP guideline-compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
TMPDE 90 (Batch 3852003), a clear, colourless liquid, was received from Perstorp on 04 September 2009 and was stored under a nitrogen headspace, protected from light, at ambient room temperature. The purity was quoted as 91.6% and the Sponsor supplied an expiry date of 03 September 2010. A Certificate of Analysis was supplied.

Method

Target gene:
tk+tk- locus
Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
The cells used were from the tk+tk- -3.7.2C mouse lymphoma L5178Y cell line obtained from Dr D Clive, Burroughs Wellcome & Company, Research Triangle Park, NC27709, USA, in December 1982. The cells grow in suspension culture, have a generation time of about 11 h, have a stable, near-diploid chromosome number and have a high cloning efficiency in serum-enriched cloning medium.
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from the liver of Aroclor 1254 induced male Fischer rats
Test concentrations with justification for top dose:
0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/mL
Vehicle:
Dimethylsulphoxide was chosen as the most appropriate solvent
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
S9 absent

Migrated to IUCLID6: 250 or 150 µg/ml
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
S9 present

Migrated to IUCLID6: 25 or 10 µg/ml
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
S9 absent

Migrated to IUCLID6: 10 or 5 µg/ml
Details on test system and conditions:
Single cultures were treated for each concentration in the initial toxicity tests. The toxicity test was performed using the standard 4 h exposure period in the absence and presence of S9 mix. An additional toxicity test was performed in the absence of S9 mix with 24 h exposure to TMPDE 90. Observations on the precipitation of TMPDE 90 were made after dosing and at the end of the exposure period. Observations of pH change (colour change in indicator in RPMI medium) were made and if any change was noted, pH measurements were made.

In the mutation assays, duplicate cultures were treated for all TMPDE 90 concentrations. On the day of the test (Day 0), samples of cell culture (in 5 mL R10P) were dispensed to sterile tubes containing 3.9 mL R0P. Freshly prepared S9 mix or R0P (1 mL) was added to each tube followed by 0.1 mL of test solution. Vehicle control cultures received 0.1 mL dimethylsulphoxide. Positive control cultures received 0.1 mL of the appropriate solution. The final reaction mixture in all cultures contained 10 mL of cells, at a population density of ca 6.0 x 105 cells/mL, in R5P medium. All tubes were incubated on a rotating drum at ca 37°C, 10 r.p.m. for 4 h. After this, the cells were gently sedimented by centrifugation at ca 200 g for 5 min and resuspended in R10P medium (20 mL). This step was repeated to give a cell density of ca 3 x 105/mL. The cells were returned to the rotating drum and allowed to express their genetic lesions at ca 37°C for 2 days. Cell numbers were adjusted, after counting, to ca 3 x 105 cells/mL on Day 1.
An experiment was conducted using an extended 24 h exposure period, in the case of results of the first experiment in the absence of S9 mix being negative. On the day of the test (Day 0), samples of cell culture (in 10 mL R10P) were dispensed to sterile tubes containing 7.8 mL R0P. R50P (R0P:serum, 50:50) (2 mL) was added to each tube followed by 0.2 mL of the test solution. Vehicle control cultures received 0.2 mL dimethylsulphoxide. Positive control cultures received 0.2 mL of the appropriate solution. The final reaction mixture in all cultures contained 20 mL of cells, at a population density of ca 3 x 105 cells/mL, in R10P medium. (The larger volumes allow the same numbers of cells to be treated as in the experiments conducted at 4 h exposure, but at half the density. The lower density is required to allow cell growth during the exposure period. The serum concentration is not lowered, as some essential nutrients can become exhausted during the exposure period.) All tubes were incubated on a rotating drum at ca 37°C, 10 r.p.m. for 24 h. After this (on Day 1), the cells were gently sedimented by centrifugation at 200 g for 5 min and resuspended in R10P medium (20 mL). This step was repeated. Cell counts were made and the densities adjusted (where higher) to give ca 3 x 105 cells/mL. The cells were returned to the rotating drum and allowed to express their genetic lesions at ca 37°C for 2 days. Cell numbers were adjusted, after counting, to ca 3 x 105 cells/mL on Day 2.
On Day 2 (4 h exposure) or Day 3 (24 h exposure), cell counts were determined. The cell counts over the 2 or 3 days of the experiments provided a measure of suspension growth. This was used when choosing dose levels to carry through to final assessment. In addition to all vehicle and positive control cultures, the cultures from the 5 highest concentrations of TMPDE 90 giving adequate cell growth were selected for assessment. The cultures were then assessed for expression of genetic damage. This was determined by performing two parallel cloning assays: the cloning efficiency assay and the mutant selection assay.

For the cloning efficiency assay, each culture was diluted into cloning medium to give an estimated 8 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 1.6 cells per well.
For the mutant selection assay, TFT stock solution was added to cloning medium to give a final concentration of 3 μg/mL. Into this medium, the cell cultures were diluted to give an estimated 1 x 104 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 2000 cells per well. All dishes were incubated at ca 37°C in an atmosphere of 5% CO2:95% air (v/v) until the colonies were fully developed (at least 9 days for cloning efficiency assay, at least 12 days for mutant selection assay).

The plates were scored using a dissecting microscope fitted to a light box with dark field illumination. The number of empty wells in each plate in the cloning efficiency assay was counted. When scoring the mutant selection assay, separate counts were made of the numbers of wells containing large type and small type colonies. Large colonies are defined as covering greater than ¼ of the floor of the well, while small colonies cover less than ¼ of the well.
Evaluation criteria:
Biological significance was assumed to apply to treatments that gave an IMF value >126 mutants per million. An experiment was considered positive if one or more concentrations were biologically significant and there was a significant linear trend. A test item was positive if 2 positive experiments out of 2 were recorded within the same activation condition.
Statistics:
Data were analysed according to the methods outlined by Robinson et al (1989). The analyses comprised the following:
1. Determination of the heterogeneity factor for each dose level.
2. Comparison of the heterogeneity factor with the historical control. Any dose levels with heterogeneity factor statistically higher than the historical control were excluded from all statistical analysis.
3. Determination of the heterogeneity factor for the experiment.
4. Calculation of a new historical control heterogeneity factor.
5. Calculation of the log mutant fraction.
6. Comparison of the log mutant fraction between the control and each treatment dose (at P<0.05).
7. Test for linear increasing trend of mutant fraction with increasing dose of test item (at P<0.05).

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
weakly positive only in the presence of S9 at toxic concentrations
Cytotoxicity:
yes
Remarks:
low to moderate toxicity without S9, higher toxicity with S9
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TMPDE 90 precipitated at the highest concentration of 5000 μg/mL; however this was of no consequence, as toxicity demanded the use of lower, non-precipitating concentrations.

When exposed to the cells for a period of 4 h in the absence of S9 mix, a concentration of 500 μg/mL reduced suspension growth to 22.1% of the vehicle control value, while 2 higher concentrations caused complete cell death. In the presence of S9 mix a concentration of 50 μg/mL reduced suspension growth to 10.8% of the vehicle control value. All higher concentrations caused complete cell death. When the exposure period in the absence of S9 mix was increased to 24 h, 150 μg/mL reduced suspension growth to 19.5% of the vehicle control, while 500 μg/mL caused near complete cell death and the 2 highest concentrations were lethal to the cells.

TMPDE 90 was assessed for mutagenic activity at concentrations of: 150, 250, 350, 450 and 550 μg/mL in the absence of S9 mix (4 h exposure). One lower concentration was surplus to requirement, while 2 higher concentrations were too toxic for assessment. None of the assessed concentrations tested significant for increase in log mutant fraction and the test for linear trend was not significant (P = 0.28). The RTG value at 550 μg/mL was 15%: a definitive level of toxicity. The experiment was classed negative, and therefore the second experiment was conducted with the extended, 24 h exposure period. In the presence of S9 mix, TMPDE 90 was assessed for mutagenic activity at concentrations of: 7.5, 12.5, 20.0, 30.0 and 42.5 μg/mL. One lower concentration was surplus to requirement, while 2 higher concentrations were too toxic for assessment. (It should be noted that the results at 20 μg/mL are based on a single replicate culture, due to a presumed dilution error in the first culture.) Evidence of a weak mutagenic response was obtained. The highest concentration of 42.5 μg/mL gave an induced mutant fraction (IMF) of 223 mutants per million. This value therefore met the IWGT criterion for a biologically relevant increase (IMF>126 mutants per million). This concentration, in addition to the 30 μg/mL treatment group, also tested significant for increase in log mutant fraction compared with the vehicle control group and the test for linear trend of mutant fraction with concentration of TMPDE 90 was highly significant (P<0.001). The RTG value at 42.5 μg/mL was 13%: a definitive level of toxicity.
TMPDE 90 was assessed for mutagenic activity at concentrations of: 40, 70, 110, 160 and 220 μg/mL in the absence of S9 mix (24 h exposure period). Two lower concentrations were surplus to requirement, while one higher concentration was too toxic for assessment. None of the assessed concentrations tested significant for increase in log mutant fraction. The test for linear trend showed a low level of significance (P = 0.020). However, all the IMF values obtained at the 4 higher concentrations were similar (25 to 28 mutants per million), and were all well below both the IWGT threshold level of 126 mutants per million. Furthermore, the IMF values were well within the normal variation for the assay (see Appendix 4, which shows that the maximum variation between pairs of vehicle control cultures over the past 5 years (no-effect maximum) for the 24 h exposure system is 49). It is therefore concluded that no meaningful increase in mutant fraction occurred in this experiment. The RTG value at 220 μg/mL was 14%: a definitive level of toxicity. In the presence of S9 mix, TMPDE 90 was assessed for mutagenic activity at concentrations of: 12.5, 20.0, 27.5, 35.0 and 42.5 μg/mL. One lower concentration was surplus to requirement, while 2 higher concentrations were too toxic for assessment. The highest concentration of 42.5 μg/mL resulted in an RTG value of 8%. As this was below the minimum acceptable value of 10%, the treatment was ruled excessively toxic and was excluded from the evaluation. Evidence of a weak mutagenic response was obtained. The highest acceptable concentration of 35.0 μg/mL gave an induced mutant fraction (IMF) of 220 mutants per million. This value therefore met the IWGT criterion for a biologically relevant increase (IMF>126 mutants per million). This concentration, in addition to the 27.5 μg/mL treatment group, also tested significant for increase in log mutant fraction compared with the vehicle control group and the test for linear trend of mutant fraction with concentration of TMPDE 90 was highly significant (P<0.001). The RTG value at 35.0 μg/mL was 15%: a definitive level of toxicity.

The mutant colony size distributions were examined for those TMPDE 90 treatments that gave a substantial increase in mutant numbers in Assays 2 and 4. Effectively, this consisted of the 42.5 μg/mL treatment in Assay 2 and the 35.0 μg/mL treatment in Assay 4. Both these treatments showed a preponderance of small type colonies (small:large ratio >1). In addition, the ratios of small to large type colonies for these treatment groups were greater than in the respective vehicle control groups. This suggests that the TMPDE 90 activity may be more closely associated with large-scale chromosomal damage, than it is with small deletions and/or point mutations.

Data from the 4 hour exposure periods are shown in Tables 1 and 2.

Any other information on results incl. tables

Table 1. Mutation test summary results without S9, 4 hour exposure

Chemical

Concentration (µg/mL)

Relative total growth (%)

Mutant fraction (x10-6)

Induced mutant fraction

(x10-6)

Ratio small to large colonies

Statistical comparison

DMSO

(100 μLadded)

100

58

N/A

1.54

N/A

EMS

250

79

450

392

0.54

*

MMS

10

37

958

901

2.09

*

TMPDE

50

NPS

NPS

NPS

NPS

NPS

150

72

53

-

1.20

NS

250

57

88

31

0.49

NS

350

42

64

6

1.25

NS

450

30

54

-

0.83

NS

550

15

90

32

1.19

NS

700

NPT

NPT

NPT

NPT

NPT

850

NPT

NPT

NPT

NPT

NPT

IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group

N/A = Not Applicable

    = Significant difference in log mutant fraction compared with vehicle control (P<0.05)

NS = Not Significant

Test for linear trend of mutant fraction with concentration of TMPDE 90 = not significant (P = 0.28)

NPS = Not Plated – Surplus

NPT = Not Plated – Toxic

-    = IMF ≤ 0

Table 2. Mutation test summary results with S9, 4 hour exposure

Chemical

Concentration (µg/mL)

Relative total growth (%)

Mutant fraction (x10-6)

Induced mutant fraction

(x10-6)

Ratio small to large colonies

Statistical comparison

DMSO

(100 μLadded)

100

64

N/A

1.23

N/A

3-MC

2.5

69

432

368

1.35

*

10

60

537

473

1.17

*

TMPDE

5.0

NPS

NPS

NPS

NPS

NPS

7.5

72

84

20

0.76

NS

12.5

54

84

20

0.82

NS

20.0

37

73

9

1.10

30.0

29

112

48

1.05

*

42.5

13

287

223

1.61

*

57.5

NPT

NPT

NPT

NPT

NPT

75.0

NPT

NPT

NPT

NPT

NPT

IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group

N/A = Not Applicable

= Significant difference in log mutant fraction compared with vehicle control (P<0.05)

NS = Not Significant

Test for linear trend of mutant fraction with concentration of TMPDE 90 = significant (P<0.001)

NPS = Not Plated – Surplus

NPT = Not Plated – Toxic

† = Results obtained from a single replicate culture only. No statistical analysis was performed.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation weakly positive with S9 only at toxic concentrations

It was concluded that TMPDE 90 is weakly mutagenic in mouse lymphoma L5178Y cells, only in the presence of S9 mix, when tested in dimethylsulphoxide at concentrations extending into the toxic range. The authors stated that while the result is insufficient evidence on its own to classify the substance as mutagenic according to GHS Category 2, it would contribute towards a Category 2 classification if supported by positive findings from relevant in vivo tests.
Executive summary:

TMPDE 90 was assayed for mutagenic potential in the mouse lymphoma L5178Y cell line, clone -3.7.2C, scoring for forward mutations at the thymidine kinase locus: tk+tk- to tk-tk-. TMPDE 90 was dissolved and diluted in dimethylsulphoxide. Tests were conducted both in the absence and in the presence of a post-mitochondrial supernatant fraction obtained from Aroclor 1254-induced livers of adult male rats and the co-factors required for mixed-function oxidase activity (S9 mix).

In preliminary cytotoxicity tests, TMPDE 90 was shown to be of a low to moderate order of toxicity in the absence of S9 mix and a higher order of toxicity in the presence of S9 mix. Exposure to a concentration of 500 μg/mL for 4 h in the absence of S9 mix caused a reduction in cell growth to 22.1% of the control value. In the presence of S9 mix a concentration of 50 μg/mL reduced relative cell growth to 10.8%. When exposure in the absence of S9 mix was extended to 24 h, a concentration of 150 μg/mL reduced relative cell growth to 19.5 %.

No relevant increase in mutant fraction was obtained with TMPDE 90 in either of the experiments in the absence of S9 mix (4h and 24h exposure). Results were obtained at concentrations resulting in a critical level of toxicity in both experiments.

Evidence of weak mutagenic activity was obtained in the presence of S9 mix. Both experiments contained one concentration that resulted in an increase in mutant fraction of greater than 126 mutants per million (the laboratory's criterion for a positive response). In addition, both experiments contained 2 treatments that tested significant for increase in log mutant fraction and both experiments tested highly significant (P<0.001) for linear trend of mutant fraction with concentration of TMPDE 90. Results meeting the laboratory's criteria for a positive response were associated with marked cytotoxicity (8 -15% RTG). Examination of the colony size distribution in the treatments giving increases >126 mutants per million showed a preponderance of small type colonies. The authors suggest that the reported activity may be associated more with large-scale chromosomal damage, than with small deletions and/or point mutations.

It was concluded that TMPDE 90 is weakly mutagenic in mouse lymphoma L5178Y cells, only in the presence of S9 mix, when tested in dimethylsulphoxide at concentrations extending into the toxic range. The authors stated that while the result is insufficient evidence on its own to classify the substance as mutagenic according to GHS Category 2, it would contribute towards a Category 2 classification if supported by positive findings from relevantin vivotests.