Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

a) Mutagenicity in vitro in bacteria; Salmonella typhimurium strains TA 1535, TA 1537, TA 1538 TA 98 and TA 100; E. coli (WP2 and WP2 uvrA) and Sacharomyces cerevisiae; negative with and without metabolic activation.

b) Gene mutation (mammalian cell gene mutation assay): HGPRT locus in V79 cells Chinese hamster: negative with and without metabolic activation (OECD 476, EPA 40, EEC 87/32)

c) Chromosome aberration test (mammalian chromosome aberration assay): V79 cell of Chinese hamster: negative with and without metabolic activation (OECD 473, EU Method B.10)

d) DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro; Freshly isolated rat hepatocytes; negative based on inability to produce a mean grain count of five or greater than the vehicle control mean grain count.

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:
1984-08-13 to 1985-01-07
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study followed standard methodology, but not all details of methodology included in report.
Qualifier:
equivalent or similar to
Guideline:
other: as described in Ames, B.N. et al, Mutation Research 31: pp. 347-364, 1975
GLP compliance:
yes (incl. certificate)
Remarks:
GLP Regulations stated in Federal Register, Vol. 43, No. 247, 12/22/1978.
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus in selected strains
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 fraction
Test concentrations with justification for top dose:
2.5 x 10-2, 5 x 10-3, 2.5 x 10-3, 5 x 10-4, 2.5 x 10-4 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: solubility of test article was best in this solvent
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
NUMBER OF REPLICATIONS: three

Evaluation criteria:
Positive response would be an increase in mutant colonies greater than three times the rate seen in the solvent control for that test strain. In the case of TA 98, the solvent control test with activation showed a rate below the spontaneous rate and the historical control. For that test, the spontaneous rate was used for comparison rather than the solvent control results.
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:
other: The highest dose rate (2.5 x 10-2 mg/plate) showed cytotoxicity for TA 1535, TA 1537, and TA 100 with and without metabolic activation.
Vehicle controls validity:
other: Solvent control data acceptable, except for TA 98 with activation. Mutation rate was below spontaneous and historical control for that test. Spontaneous rate used for comparison in that test.
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mutant colony data for Salmonella typhimurium Ames test for AN 701

Strain

Activation

Solvent Control

Positive Control

2.5 x 10-2 mg/plate

5 x 10-3 mg/plate

2.5 x 10 -3 mg/plate

5 x 10-4 mg/plate

2.5 x 10 -4 mg/plate

TA1535

-

+

6

4

35*

28*

6

4

7

5

4

5

6

8

8

8

TA1537

-

+

6

9

116*

718*

6

10

7

10

10

10

9

8

8

7

TA98

-

+

9

15**

275*

628

12

9

11

9

21

17

24

19

17

12

TA100

-

+

89

98

3175*

TNTC*

110

111

110

104

116

111

107

113

95

100

                 

* > 3 times solvent control

** Spontaneous rate

TNTC too numerous to count

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Test article did not induce a dose related increase in mutant colonies in any Salmonella typhimurium strains tested (TA 1535, TA1537, TA98, TA100) with or without metabolic activation. Therefore, AN 701 was not genetically active in the Salmonella/Microsomal Assay.
Executive summary:

Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were tested in a plate incorporation Ames test in the presence and absence of metabolic activation (S-9 fraction from livers of rats induced with Arocolor 1254) with test article dissolved in acetone. Dose levels were 2.5 x 10-2, 5 x 10-3, 2.5 x 10-3, 5 x 10-4, 2.5 x 10-4 mg AN 701/plate. An 701 was soluble at all dose levels tested. Each dose was treated in triplicate. An untreated control, solvent control, and positive control (appropriate to the strain and activation state) were tested concurrently. The highest concentration of AN 701 demonstrated toxicity in strains TA 1535, TA 1537, and TA 100 with and without metabolic activation. AN 701 did not induce a dose related increase in mutant colonies in any strains with or without activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24/06/1991-11/09/1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Chinese hamster cell line V79
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: large stocks of the V79 cell line (supplied by LMP, D-6100 Darmstadt, Germany) were stored in liquid nitrogen in the cell bank of CCR. Thawed stock cultures were propagated at 37°C in 80 cm2 plastic flasks (Greiner, D-7443 Frickenhausen, Germany). About 5 x 100000 cells were seeded into each flask with 15 ml of MEM (minimal essential medium, Seromed, D-1000 Berlin Germany), suplemented with 10% fetal calf serum (FCS, Seromed). The cells were subcultured twice weekly. The cells cultures were subcultured at 37°C and 4.5% carbon dioxide atmosphere.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
Pre-experiment:
Without and with S9 mix:
0.1, 1.0, 3.0, 6.0, 10.0, 30.0, 50.0, 100.0 µg/ml

Main test:
Without S9 mix:
18 h: 0.3, 1.0, 3.0, 4.0, 6.0, 10.0 µg/ml
28 h: 3.0, 4.0, 6.0, 10.0 µg/ml

With S9 mix:
18 h: 3.0, 10.0, 30.0, 36.0, 43.0, 50.0 µg/ml
28 h: 30.0, 36.0, 43.0, 50.0 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to cells
Untreated negative controls:
yes
Remarks:
culture medium, with and without metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, with and without metabolic activation
True negative controls:
no
Positive controls:
yes
Remarks:
two positive control substances
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: without metabolic activation, final concentration: 1.0 mg/ml = 8.0 mM
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: with metabolic activation, final concentration 4.2 µg/ml = 15 µM
Details on test system and experimental conditions:
METHOD OF APPLICATION: 3 days old logarithmically growing stock cultures more than 50% confluent were trypsinised at 37°C for approx. 5 min, then enzymatic digestion stopped by adding complete culture medium and single cell suspension was prepared. Complete medium: MEM + 10% FCS. Prior to trypsin treatment cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA. After 48 h (28 h preparation interval) and 55 h (18 h preparation interval), the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 50 µg/ml S9 mix


DURATION
- Preincubation period: 48 h (28 h preparation interval) and 55 h (18 h preparation interval), then medium was replaced with serum-free medium containing the test article, either without S9 mix or with 50 µg/ml S9 mix
- Exposure duration: 4 h. After 4 h the serum-free medium containing test article was replaced with complete medium after rinsing twice with "saline G"

- Fixation time (start of exposure up to fixation or harvest of cells): 18 h and 28 h after start of the treatment with the test article: 15.5 and 25.5 h after the start of treatment colcemid was added, 2.5 h later cells were treated with hypotonic solution for 20 min at 37°C, then cells were fixed with 3 + 1 absolute methanol + glacial acetic acid.


SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): after fixation the cells were stained with Giemsa (Merck, D-6100 Darmstadt, Germany)


NUMBER OF REPLICATIONS: the cells were seeded into Quadripem dishes which contain microscopic slides (2 chambers per dish and test group). In each chamber 5 x 10000 - 1 x 100000 cells.


NUMBER OF CELLS EVALUATED: At least 100 well spread metaphases per slide (per culture) were scored for cytogenetic damage on coded slides except the positive control cultures where 25 metaphases were scored. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis)


OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells (% polyploid metaphases, in the case of this eneuploid cell line polyploid means a near tetraploid karyotype) was scored.
- Other: chromosomal aberration


OTHER:
Acceptability of the assay:
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) the number of aberrations found in the negative and/or solvent controls fall within the laboratory historical control data range: 0.00% - 4.00%.
b) the positive control substances should reproduce significant increases in the number of cells with structural chromosome aberrations.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a concentration-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points.
A test article producing neither a concentration related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.
Statistics:
Biometry
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
mitotic index method
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment on toxicity (colony forming ability) in the absence and presence of S9 mix after treatment with concentrations higher than 3.0 (without S9 mix) and 30.0 µg/ml (with S9 mix) the colony forming ability was distinctly reduced.




Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Pre-experiment for toxicity:

Table 1: Plating Efficiency Assay (PE) without metabolic activation. Per flask 495 cells were seeded

Conc. Per ml

Colonies counted

mean

PE% relative

Flask I

Flask II

Negative control

301

248

274.5

 

Solvent control (DMSO)

280

271

275.5

100.0

0.1µg

266

291

278.5

101.1

1.0µg

221

259

240.0

87.1

3.0µg

288

300

294.0

106.7

6.0µg

2

9

5.5

2.0

10.0µg

0

0

0.0

0.0

30.0µg

0

0

0.0

0.0

50.0µg

0

0

0.0

0.0

100µg

0

0

0.0

0.0

Table 2: Plating Efficiency Assay (PE) with metabolic activation. Per flask 495 cells were seeded

Conc. Per ml

Colonies counted

mean

PE% relative

Flask I

Flask II

Negative control

309

309

309.0

 

Solvent control (DMSO)

299

271

285.0

100.0

0.1µg

321

287

304.0

106.7

1.0µg

313

281

297.0

104.2

3.0µg

282

302

292.0

102.5

6.0µg

301

299

300.0

105.3

10.0µg

256

284

270.0

94.7

30.0µg

320

279

299.5

105.1

50.0µg

0

0

0.0

0.0

100µg

0

0

0.0

0.0

Main study: In the main experiment the mitotic index was reduced after treatment with the highest scorable concentration in the absence and presence of S9 mix except at interval 28 h in the presence of S9 mix. Both, in the absence and presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval to a biologically relevant extend. The aberration rates of the cells after treatment with the test article (0.5% - 3.0%) were in or near to the range of the control values: 0.0% - 2.0%). At fixation interval 18h, in the absence of S9 mix after treatment with 10.0 µg/ml, 3 cells were observed carrying exchanges and no cells with exchanges in the corresponding solvent control. However, at interval 28h a similar number of exchanges (2) were found in the control cultures (with S9 mix). Therefore, this effect observed in the samples treated with the test article can be regarded as being biologically not relevant. Due to relative high toxicity found in one of the replicates in this test group an additional sample were evaluated treated with 6.0 µg/ml. In this sample, no evidence of a clastogenic potential was observed. Table 4 shows the occurrence of polyploid metaphases. No relevant deviation from the control data was found after treatment with the test article.

EMS (1.0 mg/ml) nad CPA (4.2 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Table 3a: mitotic index (18 h)

Test group

Conc. Per ml

S9 mix

Fixation interval (h)

Mitotic index per cent*

slide

Abs.

Rel.

1

2

Negative control

0.0µg

-

18

21.0

19.2

20.1

100.0

Solvent control DMSO

1.0%

-

18

16.1

13.3

14.7

100.0

 Positive control EMS

1.0 mg

-

18

12.3

13.3

12.8

63.8

Test article

0.3µg

-

18

26.8

18.6

22.7

154.4

Test article

1.0µg

-

18

14.6

17.4

16.0

108.8

Test article

3.0µg

-

18

17.1

19.6

18.4

125.2

Test article

4.0µg

-

18

14.8

14.4

14.6

99.3

Test article

6.0µg

-

18

5.7

5.7

5.7

38.8

Test article

10.0µg

-

18

7.4

0.0

3.7

25.2

Negative control

0.0µg

+

18

18.4

19.2

18.8

100.0

Solvent control DMSO

1.0 %

+

18

16.2

18.8

17.5

100.0

 Positive control CPA

4.2µg

+

18

4.4

8.6

6.5

34.6

Test article

3.0µg

+

18

21.4

13.4

17.4

99.4

Test article

10.0µg

+

18

11.7

28.7

20.2

115.4

Test article

30.0µg

+

18

26.2

19.3

22.8

130.3

Test article

36.0µg

+

18

18.3

8.9

13.6

77.7

Test article

43.0µg

+

18

14.2

14.8

14.5

82.9

Test article

50.0µg

+

18

1.6

5.0

3.3

18.9

* the mitotic index was determined in 1000 cells from each of the two slides per test group

Table 3b: mitotic index (28 h)

Test group

Conc. Per ml

S9 mix

Fixation interval (h)

Mitotic index per cent*

slide

Abs.

Rel.

1

2

Solvent control DMSO

1.0%

-

28

14.9

13.3

14.6

100.0

Test article

3.0µg

-

28

10.0

13.5

11.8

61.0

Test article

4.0µg

-

28

6.3

12.2

9.3

63.7

Test article

6.0µg

-

28

9.0

5.6

7.3

79.5

Test article

10.0µg

-

28

0.0

1.9

1.0

6.8

Solvent control DMSO

1.0 %

+

28

13.5

10.9

12.2

100.0

Test article

30.0µg

+

28

14.0

16.6

15.3

96.7

Test article

36.0µg

+

28

10.2

9.6

9.9

81.1

Test article

43.0µg

+

28

13.5

14.3

13.9

113.9

Test article

50.0µg

+

28

13.0

13.4

13.2

108.2

* the mitotic index was determined in 1000 cells from each of the two slides per test group

Table 4: number of polyploid cells

Test group

Conc. Per ml

S9 mix

Fixation interval (h)

Polyploid cells*

slide

Total

mean

1

2

Negative control

0.0µg

-

18

1.0

3.0

4.0

2.0

Solvent control DMSO

1.0%

-

18

5.0

4.0

9.0

4.5

 Positive control EMS

1.0 mg

-

18

3.0

2.0

5.0

2.5

Test article

0.3µg

-

18

2.0

6.0

8.0

4.0

Test article

3.0µg

-

18

4.0

3.0

7.0

3.5

Test article

6.0µg

-

18

2.0

6.0

8.0

4.0

Test article

10.0µg

-

18

6.0

n.s.

6.0

6.0

Negative control

0.0µg

+

18

3.0

2.0

5.0

2.5

Solvent control DMSO

1.0 %

+

18

0.0

4.0

4.0

2.0

Positive control CPA

4.2µg

+

18

2.0

1.0

3.0

1.5

Test article

3.0µg

+

18

1.0

6.0

7.0

3.5

Test article

30.0µg

+

18

1.0

1.0

2.0

1.0

Test article

50.0µg

+

18

12.0

2.0

4.0

2.0

Solvent control

0.0µg

-

28

4.0

5.0

9.0

4.5

Test article

6.0µg

-

28

5.0

7.0

12.0

6.0

Solvent control

0.0µg

+

28

1.0

4.0

5.0

2.5

Test article

50.0µg

+

28

3.0

3.0

6.0

3.0

*the number of polyploidy cells was determined in 100 cells from each of the two slides per test group.

n.s. = not scorable due to low metaphase number

Table 21: Mutagenicity data

Fixation interval: 18 h after start of treatment

Test group

Number of cells analysed

Conc. Per ml (µg)

 

S9 mix

Per cent aberrant cells

incl. gaps

Excl. gaps

exchanges

Negative control

200

0.0

-

2.00

1.00

0.00

Solvent control

200

0.0

-

2.00

1.00

0.00

 Positive control EMS

50

1.0

-

16.00

14.00

10.00

Test article

200

0.3

-

1.50

0.50

0.50

Test article

200

3.0

-

1.00

1.00

1.00

Test article

200

6.0

-

1.50

0.50

0.00

Test article

200*

10.0

+

4.00

3.00

1.50

Negative control

200

0.0

+

2.50

0.50

0.00

Solvent control

200

0.0

+

3.50

1.50

0.50

 Positive control CPA

50

4.2

+

42.00

42.00

20.00

Test article

200**

3.0

+

0.50

0.50

0.00

Test article

200

30.0

+

1.50

1.00

0.00

Test article

200

50.0

+

3.50

1.50

0.50

*200 metaphases were scored on slide 1 due to low metaphase number on slide 2

** 200 metaphases were scored on slide 1 due to poor spreading quality of metaphases on slide 2

 

Table 22: Mutagenicity data

Fixation interval: 28 h after start of the treatment

Test group

Number of cells analysed

Conc. Per ml (µg)

 

S9 mix

Per cent aberrant cells

incl. gaps

Excl. gaps

exchanges

Solvent control

200

0.0

-

2.00

0.00

0.00

Test article

200

6.0

-

3.00

0.50

0.50

Solvent control

200

0.0

+

4.00

2.00

1.00

Test article

200

50.0

+

1.00

1.00

0.50

Conclusions:
Interpretation of results (migrated information):
negative non-mutagenic in this chromosomal aberration test

In conclusion it can be stated that in the study described and under the experimental conditions reported, the test article did not induced structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, 2,6-bis(1,1-dimethylethyl)-phenol is considered to be non-mutagenic in this chromosomal aberration test.
Executive summary:

The test article 2,6 -bis(1,1 -dimethyethyl)-phenol was assessed for its potential to induce structural chromosome aberrations in V79 cell of Chinese hamster in vitro.

Preparation of chromosomes was done 18 h (low, medium and high concentration range), and 28 h (high concentration range) after start of treatment with the test article. The treatment interval was 4 h.

In each experiment group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations except the positive control cultures were 25 metaphases were scored.

The following concentrations were evaluated:

Without S9 mix:

18 h: 0.3, 3.0, 6.0, 10.0 µg/ml

28 h: 6.0 µg/ml

With S9 mix:

18 h: 3.0, 30.0, 50.0 µg/ml

28 h: 50.0 µg/ml

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response.

Treatment with concentrations higher than 3.0 (without S9 mix) and 30.0 mg/ml (with S9 mix) reduced distinctly the plating efficiency of the V79 cells. Also the mitotic index was reduced in the samples treated with the highest scorable concentration at both fixation intervals except at interval 28 h in the presence of S9 mix.

There was no relevant increase in cells with structural aberrations after treatment with the test article at each fixation interval either without or with metabolic activation by S9 mix.

Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion it can be stated that in the study described and under the experimental conditions reported, the test article did not induced structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, 2,6-bis(1,1-dimethylethyl)-phenol is considered to be non-mutagenic in the chromosomal aberration test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15/07/1991-19/08/1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, 40 CFR, Ch.I, part 798, Detection of gene mutation in somatic cells in culture, pp. 717-720 (7-1-86 Edition)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Directive 87/302, L 133, p. 61-63
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: large stocks of the V79 cell line (supplied by LMP, D-6100 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of CCR. Thawed stock cultures are propagated at 37°C in 80 cm2 plastic flasks (Greiner, D-7440 Nürtingen, Germany). About 5 x 100000 cells are seeded into each flask with 15 ml of MEM (minimal essential medium, Seromed, D-1000 Berlin germany), supplemented with 10% foetal calf serum (FCS, Seromed). The cells are subcultured twice weekly. The cells cultures are incubated at 37°C and 4.5% carbon dioxide atmosphere. For the selection of mutants the medium is supplemented with 11 µg/ml thioguanine (Sigma GmbH, D-8024 Deisenhofen, Germany)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
liver microsome preparations
Test concentrations with justification for top dose:
Pre-experiment for toxicity (colony forming ability):
- Without and with metabolic activation 0.10, 1.00, 3.00, 6.00, 10.00, 30.00, 50.00, 100 µg

Main experiments:
- Without metabolic activation: 0.30, 1.00, 2.00, 4.00, 6.00, 8.00 µg/ml
- With metabolic activation: 3.00, 10.00, 20.00, 30.00, 40.00, 50.00 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO. On the day of the experiment the test article was dissolved in DMSO (Merck, D-6100 Darmstadt, Germany)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to cells.
Untreated negative controls:
yes
Remarks:
concurrent
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
two positive control substances
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: Without metabolic activation, final concentration 0.6 mg/ml = 4.8 mM
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Migrated to IUCLID6: With metabolic activation, final concentration 3.85 µg/ml = 15.03 µM
Details on test system and experimental conditions:
METHOD OF APPLICATION: 2 or 3 days old logarithmically growing stock cultures more than 50% confluent were trypsinized at 37°C for 5 min. Then digestion stopped by adding complete medium and a single cell suspension prepared. Prior to trypsin treatment cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA. The cell suspension was seeded into plastic culture flasks (Greines, D-7440 Nürtingen, Germany). Approx. 8 x 100000 and 5 x 100 cells, respectively, were seeded in each flask. The medium was MEM with 10% FCS (complete medium)


DURATION
- Preincubation period: After 24 h the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 50 µl/ml S9 mix.
- Exposure duration: After 4 h the treatment medium was replaced with normal medium after rinsing twice with "saline G".

Experimental Scheme:

Segment a) Procedure for determination of toxicity
Segment b) Procedure for determination of mutation rate

Day 1: subculturing of a log-phase culture
a) About 500 cells in 5 ml medium/25 cm2-plastic-flask for plating efficiency; in duplicate per experimental point.
b) 8 x 100000 cells in 30 ml medium/175 cm2-plastic flask for the mutagenicity test, 1 flask per experimental poit

Day 2: treatement of a) and b)

Experiment 1
Day 5: subculturing of b) in 175 cm2-plastic-flasks or
experiment 2
Day 6: see day 5

Day 8: fixation and staining of colonies in a)-flasks = plating efficiency and concentration relationship

Day 9: subculturing of b) in five 80 cm2-plastic-flasks containing selective medium: mutant selection (about 3-5 x 100000 cell/flask); subculturing of b) in two 25 cm2-flasks for plating efficiency (about 500 cells/flask)

Day 16: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (plating efficiency)

Day 17: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (mutant selection)


SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): 10 % methylene blue in 0.01 % KOH solution (E. Merck, D-6100 Darmstadt, Germany). The stained colonies with more than 50 cells were counted with a preparation microscope (Nikon, D-4000 Düsseldorf, Germany)


Evaluation criteria:
A test article is classified positive if it induces either a significant concentration-related increase in the mutant frequency or a reproducible and significant positive response for at least one of the test points.

A test article producing neither a significant concentration related increase in the mutant frequency nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
The test article is classified as mutagenic if it induces reproducibly with at least one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment.
The test article is classified as mutagenic if there is a reproducible concentration - related increase in the mutation frequency. Such evaluation may be considered independently of an enhancement factor for induced mutants.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate as compared to the range normally found (5-45 mutants per 1000000 cells) a seemingly concentration-related increase of the mutations within this range will be regarded as to be irrelevant.
Statistics:
Biometry
Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
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'.

Pre-experiment for toxicity

(the results were obtained from CCR Project 243628, Chromosome aberration)

Plating Efficiency Assay without metabolic activation, 495 single cells were seeded into each flask

Conc. Per ml

Colonies counted

mean

PE% relative

Flask I

Flask II

Negative control

301

248

274.5

 

Solvent control (DMSO)

280

271

275.5

100.0

0.1µg

266

291

278.5

101.1

1.0µg

221

259

240.0

87.1

3.0µg

288

300

294.0

106.7

6.0µg

2

9

5.5

2.0

10.0µg

0

0

0.0

0.0

30.0µg

0

0

0.0

0.0

50.0µg

0

0

0.0

0.0

100µg

0

0

0.0

0.0

Plating Efficiency Assay with metabolic activation, 495 single cells were seeded into each flask

onc. Per ml

Colonies counted

mean

PE% relative

Flask I

Flask II

Negative control

309

309

309.0

 

Solvent control (DMSO)

299

271

285.0

100.0

0.1µg

321

287

304.0

106.7

1.0µg

313

281

297.0

104.2

3.0µg

282

302

292.0

102.5

6.0µg

301

299

300.0

105.3

10.0µg

256

284

270.0

94.7

30.0µg

320

279

299.5

105.1

50.0µg

0

0

0.0

0.0

100µg

0

0

0.0

0.0

The test article was tested in experiment I (without S9 mix) and II (with S9 mix).

In the first experiment the plating efficiency was reduced at the highest concentration but in the second experiment no decrease of the plating efficiency was observed up to the highest concentration (see tables I and IV), PE% relative).

Table I: toxicity data, experiment I

Column

Conc. Per ml

S9 mix

Number of cells per flask*

PE%** absolute

PE%*** relative

seeded

found

mean

I/II

I

II

 

1

2

3

4

5

6

7

8

Negative control

0.00µg

-

497

298

299

298.5

60.1

100.0

Solvent control DMSO

0.00µg

-

497

260

260

260.0

52.3

100.0

 Positive control EMS

0.60µg

-

497

197

160

178.5

35.9

59.8

Test article

0.30µg

-

497

266

257

261.5

52.6

100.6

Test article

1.00µg

-

497

359

232

245.5

49.4

94.4

Test article

2.00µg

-

497

258

252

255.0

51.3

98.1

Test article

4.00µg

-

497

309

272

290.5

58.5

111.7

Test article

6.00µg

-

497

220

226

223.0

44.9

85.8

Test article

8.00 µg

-

497

13

20

16.5

3.3

6.3

Negative control

0.00µg

+

497

307

305

306.0

61.6

100.0

Solvent control DMSO

0.00µg

+

497

246

248

247.0

49.7

100.0

Positive control DMBA

3.85µg

+

497

270

249

259.5

52.2

105.1

Test article

3.00µg

+

497

256

271

263.5

53.0

86.1

Test article

10.00µg

+

497

227

234

230.5

46.4

75.3

Test article

20.00µg

+

497

253

253

253.0

50.9

82.7

Test article

30.00µg

+

497

244

259

251.5

50.6

82.2

Test article

40.00µg

+

497

258

299

278.5

56.0

91.0

Test article

50.00µg

+

497

217

240

228.5

46.0

74.7

*only colonies with more than 50 cells 7 days after seeding were scored

** PE absolute (value column 6/value column 3 x 100)

***PE relative (value column 6/value column 6 of corresponding control x 100)

Table II: Mutagenicity data, experiment I (part 1: cell survival)

Column

Conc. Per ml

S9 mix

Number of cells per flask*

Factor** calculated

Cells seeded

Cells*** survived

seeded

found

mean

 

 

 

 

I/II

I

II

 

1

2

3

4

5

6

7

8

9

Negative control

0.00µg

-

556

402

367

384.5

0.69

387000

267030

Negative control DMSO

0.00µg

-

474

391

432

411.5

0.87

453000

394110

 Positive control EMS

0.60µg

-

567

457

431

444.0

0.78

375000

292500

Test article

0.30µg

-

486

380

410

395.0

0.81

450000

364500

Test article

1.00µg

-

526

300

335

317.5

0.60

480000

288000

Test article

2.00µg

-

Culture was not continued

 

 

 

Test article

4.00µg

-

539

321

333

327.0

0.61

426000

259860

Test article

6.00µg

-

Culture was not continued

 

 

 

Test article

8.00 µg

-

530

335

363

349.0

0.66

372000

245520

Negative control

0.00µg

+

568

380

373

376.5

0.66

450000

297000

Negative control DMSO

0.00µg

+

537

357

344

350.5

0.65

432000

280800

 Positive control DMBA

3.85µg

+

479

307

323

315.0

0.66

423000

279180

Test article

3.00µg

+

579

374

331

352.5

0.61

402000

245220

Test article

10.00µg

+

461

324

330

327.0

0.71

450000

319500

Test article

20.00µg

+

Culture was not continued

 

 

Test article

30.00µg

+

497

316

315

315.5

0.63

435000

274050

Test article

40.00µg

+

Culture was not continued

 

 

 

Test article

50.00µg

+

486

264

297

280.5

0.58

435000

252300

*only colonies with more than 50 cells 7 days after seeding in normal medium were scored

** factor calculated (value column 6/value column 3)

***cells survived after plating in TG containing medium (value column 8 x value column 7)

Table III: Mutagenicity data, experiment I (part 2: mutation rates)

Column

Conc per ml

S9 mix

Number of mutant colonies per flask* found after plating in TG medium

Standard deviation

Mutant** colonies per 106cells

I

II

III

IV

V

mean

1

2

3

4

5

6

7

8

9

10

Negative control

0.00µg

-

7

1

11

6

4

5.8

3.7

21.7

Negative control DMSO

0.00µg

-

5

1

3

4

3

3.2

1.5

8.1

 Positive control EMS

0.60µg

-

103

83

93

91

80

90.0

9.1

307.7

Test article

0.30µg

-

14

16

18

11

8

13.4

4.0

36.8

Test article

1.00µg

-

3

6

7

4

10

6.0

2.7

20.8

Test article

2.00µg

-

Culture was not continued

 

 

 

 

Test article

4.00µg

-

4

8

8

6

9

7.0

2.0

26.9

Test article

6.00µg

-

Culture was not continued

 

 

 

 

Test article

8.00 µg

-

10

3

6

4

2

5.0

3.2

20.4

Negative control

0.00µg

+

5

8

5

8

11

7.4

2.5

24.9

Negative control DMSO

0.00µg

+

14

11

18

11

15

13.8

2.9

49.1

 Positive control DMBA

3.85µg

+

131

123

126

139

101

124.0

14.2

444.2

Test article

3.00µg

+

4

10

5

12

10

8.2

3.5

33.4

Test article

10.00µg

+

5

6

8

3

5

5.4

1.8

16.9

Test article

20.00µg

+

Culture was not continued

 

 

 

 

Test article

30.00µg

+

6

5

6

8

8

6.6

1.3

24.1

Test article

40.00µg

+

Culture was not continued

 

 

 

 

Test article

50.00µg

+

5

9

8

11

5

7.6

2.6

30.1

*only colonies with more than 50 cells 8 days after seeding in TG medium were scored

**value column 8 x106/value column 9 (table I)

Table IV: toxicity data, experiment II

Column

Conc. Per ml

S9 mix

Number of cells per flask*

PE%** absolute

PE%*** relative

seeded

found

mean

I/II

I

II

 

1

2

3

4

5

6

7

8

Negative control

0.00µg

-

497

295

296

295.5

59.5

100.0

Solvent control DMSO

0.00µg

-

497

289

262

275.5

55.4

100.0

 Positive control EMS

0.60µg

-

497

291

240

265.5

53.4

89.8

Test article

0.30µg

-

497

353

277

315.0

63.4

114.3

Test article

1.00µg

-

497

346

342

344.0

69.2

124.9

Test article

2.00µg

-

497

280

287

283.5

57.0

102.9

Test article

4.00µg

-

497

276

322

299.0

60.2

108.5

Test article

6.00µg

-

497

371

341

356.0

71.6

129.2

Test article

8.00 µg

-

497

310

315

312.5

62.9

113.4

Negative control

0.00µg

+

497

325

314

319.5

64.3

100.0

Solvent control DMSO

0.00µg

+

497

317

297

307.0

61.8

100.0

Positive control DMBA

3.85µg

+

497

191

206

198.5

39.9

64.7

Test article

3.00µg

+

497

303

284

293.5

59.1

91.9

Test article

10.00µg

+

497

315

267

291.0

58.6

91.1

Test article

20.00µg

+

497

299

297

298.0

60.0

93.3

Test article

30.00µg

+

497

289

303

296.0

59.6

92.6

Test article

40.00µg

+

497

282

272

277.0

55.7

86.7

Test article

50.00µg

+

497

263

294

278.5

56.0

87.2

*only colonies with more than 50 cells 7 days after seeding were scored

** PE absolute (value column 6/value column 3 x 100)

***PE relative (value column 6/value column 6 of corresponding control x 100)

Table V: mutagenicity data experiment II (part 1: cell survival)

Column

Conc. Per ml

S9 mix

Number of cells per flask*

Factor** calculated

Cells seeded

Cells*** survived

seeded

found

mean

 

 

 

 

I/II

I

II

 

1

2

3

4

5

6

7

8

9

Negative control

0.00µg

-

487

512

452

482.0

0.99

392000

388080

Negative control DMSO

0.00µg

-

450

367

420

393.5

0.87

435000

378450

 Positive control EMS

0.60µg

-

515

434

448

441.0

0.86

444000

381840

Test article

0.30µg

-

497

398

400

399.0

0.80

426000

340800

Test article

1.00µg

-

476

375

401

388.0

0.82

498000

408360

Test article

2.00µg

-

Culture was not continued

 

 

 

Test article

4.00µg

-

452

386

421

403.5

0.89

492000

437880

Test article

6.00µg

-

Culture was not continued

 

 

 

Test article

8.00 µg

-

480

446

460

453.0

0.94

435000

408900

Negative control

0.00µg

+

505

377

397

387.0

0.77

369000

284130

Negative control DMSO

0.00µg

+

492

338

436

387.0

0.79

306000

241740

 Positive control DMBA

3.85µg

+

494

333

334

333.5

0.68

396000

269280

Test article

3.00µg

+

495

369

393

381.0

0.77

402000

309540

Test article

10.00µg

+

510

346

308

327.0

0.64

378000

241920

Test article

20.00µg

+

Culture was not continued

 

 

Test article

30.00µg

+

533

245

283

264.0

0.50

387000

193500

Test article

40.00µg

+

Culture was not continued

 

 

 

Test article

50.00µg

+

493

407

367

387.0

0.78

384000

299520

*only colonies with more than 50 cells 7 days after seeding in normal medium were scored

** factor calculated (value column 6/value column 3)

***cells survived after plating in TG containing medium (value column 8 x value column 7)

See Table VI in overall remarks.

Taking into account the mutation rates found in the groups treated with the test article compared to the negative and solvent controls it can be concluded that no biologically relevant increase of point mutations was observed. The test article did not induce reproducible concentration-related increase in the mutant colony numbers. The mutant values of the groups treated with the test article were in the range of the negative controls.

In this study in both experiment (with and without S9 mix) the range of the negative controls was from 0.7 up to 49.1 mutants per 1000000 cells, the range of the groups treated with the test article was from 0.5 up to 36.8 mutants per 1000000 cells.

EMS (0.6 mg(ml) and DMBA (3.85 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:
Interpretation of results (migrated information):
negative non mutagenic in this HGPRT assay

In conclusion can be stated that during the described mutagenicity test and under the experimental conditions reported the test article did not induce point mutations at the HGPRT locus in V79 cells.
Executive summary:

The study was performed to investigate the potential of 2,6 -bis(1,1 -dimethyethyl)-phenol to induce gene mutations at the HGPRT locus in V79 cells of the Chinese hamster in vitro.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation.

The test article was tested with the following concentrations:

Experiment I and II:

Without S9 mix: 0.3, 1 -0, 2.0, 4.0, 6.0*, and 8.0 µg/ml

With S9 mix: 3.0, 10.0, 20.0*, 30.0, 40.0, 50.0 µg/ml

* culture was not continued in experiment I and II

According to the pre-experiment for toxicity the concentration ranges were selected to yield concentration related toxic effects. In the first experiment the highest concentration produced a low level of survival and the survival at the lowest concentration was approximately in the range of the negative control.

At the highest investigated concentration no relevant increase in mutant colony numbers was obtained in two independent experiments.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that during the described mutagenecity test and under the experimental conditions reported the test article did not induce point mutations at the HGPRT locus in V79 cells.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro:

Mutagenicity in vitro in bacteria

Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were tested in a plate incorporation Ames test (in the presence and absence of metabolic activation (S-9 fraction from livers of rats induced with Arocolor 1254) with 2,6-di-tert-butylphenol (AN 701) dissolved in acetone. Dose levels were 2.5 x 10-2, 5 x 10-3, 2.5 x 10-3, 5 x 10-4, 2.5 x 10-4mg AN 701/plate. AN 701 was soluble at all dose levels tested. Each dose was treated in triplicate. An untreated control, solvent control, and positive control (appropriate to the strain and activation state) were tested concurrently. The highest concentration of AN 701 demonstrated toxicity in strains TA 1535, TA 1537, and TA 100 with and without metabolic activation. AN 701 did not induce a dose related increase in mutant colonies in any strains with or without activation.

Test method and results:

The bacterial reverse mutation assay (Ames test) was conducted as described in Ames, B.N. et al, Mutation Research 31: pp. 347-364, 1975, at the following concentration levels:-

2.5 x 10-2, 5 x 10-3, 2.5 x 10-3, 5 x 10-4, 2.5 x 10-4mg AN 701/plate

Species/strain -S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation -with and without

Test system -all strains/cell types tested

Genotoxicity -negative

Cytotoxicity -other: The highest dose rate (2.5 x 10-2 mg/plate) showed cytotoxicity for TA 1535, TA 1537, and TA 100 with and without metabolic activation.

Vehicle controls valid -other: Solvent control data acceptable, except for TA 98 with activation. Mutation rate was below spontaneous and historical control for that test. Spontaneous rate used for comparison in that test.

Positive controls valid -yes

Test article did not induce a dose related increase in mutant colonies in any Salmonella typhimurium strains tested (TA 1535, TA1537, TA98, TA100) with or without metabolic activation. Therefore, AN 701 was not genetically active in the Salmonella/Microsomal Assay.

Gene mutation (mammalian cell gene mutation assay)

The study was performed to investigate the potential of 2,6 -bis(1,1 -dimethyethyl)-phenol to induce gene mutations at the HGPRT locus in V79 cells of the Chinese hamster in vitro. In conclusion it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations at the HGPRT locus in V79 cells.

Test method and results:

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation.

The test article was tested with the following concentrations:

Experiment I and II:

Without S9 mix: 0.3, 1.0, 2.0, 4.0, 6.0*, and 8.0 µg/ml

With S9 mix: 3.0, 10.0, 20.0*, 30.0, 40.0, 50.0 µg/ml

* culture was not continued in experiment I and II

According to the pre-experiment for toxicity the concentration ranges were selected to yield concentration related toxic effects. In the first experiment the highest concentration produced a low level of survival and the survival at the lowest concentration was approximately in the range of the negative control.

Up to the highest investigated concentration no relevant increase in mutant colony numbers was obtained in two independent experiments.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.

Chromosome aberration test (mammalian chromosome aberration assay):

The test article 2,6 -bis(1,1-dimethyethyl)-phenol was assessed for its potential to induce structural chromosome aberrations in V79 cell of Chinese hamster in vitro. In conclusion it can be stated that in the study described and under the experimental conditions reported, the test article did not induced structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, 2,6-bis(1,1-dimethylethyl)-phenol is considered to be non-mutagenic in this chromosomal aberration test.

Test method and results:

Preparation of chromosomes was done 18 h (low, medium and high concentration range), and 28 h (high concentration range) after start of treatment with the test article. The treatment interval was 4 h.

In each experiment group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations except the positive control cultures where 25 metaphases were scored.

The following concentrations were evaluated:

Without S9 mix:

18 h: 0.3, 3.0, 6.0, 10.0µg/ml

28 h: 6.0 µg/ml

With S9 mix:

18 h: 3.0, 30.0, 50.0 µg/ml

28 h: 50.0 µg/ml

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response.

Treatment with concentrations higher than 3.0 (without S9 mix) and 30.0 mg/ml (with S9 mix) reduced distinctly the plating efficiency of the V79 cells. Also the mitotic index was reduced in the samples treated with the highest scorable concentration at both fixation intervals except at interval 28 h in the presence of S9 mix.

There was no relevant increase in cells with structural aberrations after treatment with the test article at each fixation interval either without or with metabolic activation by S9 mix.

Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Freshly isolated rat hepatocytes were treated with 20 µl test article solution per 2 ml media to give concentrations of AN 701 (2,6 -DTBP) of 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, and 1000 µg/well. In addition a solvent control (acetone), an untreated control, and a positive control (2 -acetoamidofluorene at 10 -6M) were assessed concurrently. Cytotoxicity was seen at AN 701 concentrations of 50 µg/well and higher. The highest dose scored for AN 701 was 10 µg/ml. Unscheduled DNA repair synthesis was quantified by a net nuclear increase of black silver grains for 25 cells per slide. This value was determined by taking a nuclear count and the average of three adjacent cytoplasmic counts. AN 701 did not cause an increase in mean net nuclear counts over solvent counts at any dose level counted. The number of cells with net nuclear grain counts greater than zero of all scored dose levels was not significantly increased statistically over solvent control. Under the conditions of the test, AN 701 was negative in the Rat Hepatocyte Primary Culture/DNA Repair test based on inability to produce a mean grain count of five or greater than the vehicle control mean grain count, and no statistically increased number of cells with nuclear grain count greater than zero compared to vehicle control.

 

Test method and results:

Male Fischer 344 rats were obtained from Charles River Breeding Laboratories, and weighed from 150 to 300 grams at the start of the study. Animals were housed at the Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana. For recovery of the hepatocytes for the test, the rats were anesthetized by sodium Nembutal injection intraperitoneally. When anesthetized, the liver was perfused with Medium A (0.5 mM EDTA) and Medium B (Collagenase, 100 units/mL), and the livers removed, and the hepatocytes harvested. Freshly isolated rat hepatocytes were then treated with 20 ul of AN 701 at doses of 0.05, 0.1, 0.5, 1, 10, 50, 100, 500, and 1000 ug/well in 2 mL of media. Additionally, an acetone group, an untreated control and a positive control (2 -acetoamidofluorene, 2 -AAF at 10 -6M) were concurrently evaluated. After treatment, hepatocytes were fixed on microscope slides, stained, dipped and developed for assessment of black silver grains in the nuclei. An automatic colony counter was used to evaluate 25 cells for grain counts in the nuclei. Net nuclear count value was determined by taking a nuclear count and the average of three adjacent cytoplasmic counts.

AN 701 treatment did not cause an increase in mean net nuclear count over solvent for any dose level counted. Cytotoxicity was produced at levels of 50 ug/well and higher. Thus, the highest level scored was 10 ug/well.

Waiving argument against an additional Ames-Test with 5 strains

Data basis

The following in vitro genotoxicity tests are currently available in the REACH Registration Dossier of 2,6-bis(1,1-dimethylethyl)-phenol (CAS No 128-39-2, EC No 204-884-0, registration number: 01-2119490822-33-0000) (see http://echa.europa.eu/information-onchemicals/registered-substances):

Genetic toxicity in vitro:

a) Mutagenicity in vitro in bacteria; Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100; negative with and without metabolic activation.

b) Gene mutation (mammalian cell gene mutation assay): HGPRT locus in V79 cells Chinese hamster: negative with and without metabolic activation (OECD 476, EPA 40, EEC 87/32)

c) Chromosome aberration test (mammalian chromosome aberration assay): V79 cell of Chinese hamster: negative with and without metabolic activation (OECD 473, EU Method B.10)

d) DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro; Freshly isolated rat hepatocytes; negative based on inability to produce a mean grain count of five or greater than the vehicle control mean grain count.

Scientific analysis

The Ames test was conducted in 1985 and was in full compliance to the relevant guideline OECD 471 at that date including GLP compliance. As some details on the methodology were missing in the report a reliability score of 2 was assigned to the test and as the consortium understands, this assignment is not questioned by ECHA. The test reports negative results for all four test strains.

A supporting study is available in literature (Dean et al., 1985), in which 2 -DTBP was tested in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, E. coli (WP2 and WP2 uvrA) and Sacharomyces cerevisae were tested in a pre-incubation and plate incorporation Ames test in the presence and absence of metabolic activation (S-9 fraction from livers of rats induced with Arocolor 1254) with test article dissolved in DMSO. Dose levels were 125, 250, 500, 1000, 2000 and 4000 µg/plate. Each dose was treated at least in triplicate. A solvent control, and positive control (appropriate to the strain and activation state) were tested concurrently. The test item did not induce an increase in mutant colonies in any strains with or without activation.

Conclusion

Firstly in compliance with the standard data requirements a reliable in vitro mammalian cell gene mutation assay and a reliable in vitro chromosome aberration assay are available. As a surplus data from a reliable UDS assay (unscheduled DNA synthesis) in mammalian cells in vitro are presented in the REACH Registration Dossier for 2,6-bis(1,1-dimethylethyl)-phenol (CAS No 128-39-2, EC No 204-884-0, registration number: 01-2119490822-33-0000). It is the consortium's understanding that the reliability of these tests is not questioned by ECHA.

Both the in vitro mammalian cell gene mutation assay as well as the in vitro UDS assay in mammalian cells aim at the same endpoint as the Ames test, which is gene mutation. We regard it as extremely unlikely that the combination of these two test systems is not able to detect a potential genotoxic mechanism that is relevant to humans that could on the other hand be detected by E.coli WP2 strains or S. typhimurium TA102 in the Ames tests. Especially the surplus in vitro UDS assay in mammalian cells is in our eyes very well suited to cover the information gap that stems from the lacking 5th strain in the available Ames test.

In the Guidance on information requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint specific guidance, Version 2.2, August 2013) it is stated: "If negative results are available from an adequate evaluation of genotoxicity from existing data in appropriate test systems, there may be no requirement to conduct additional genotoxicity tests." With regard to the available four reliable in vitro genotoxicity tests the consortium has the opinion that this requirement is fulfilled and that no additional testing is required in order to allow a solid hazard assessment for the genotoxicity potential of 2,6-bis(1,1-dimethylethyl)-phenol (CAS No 128-39-2, EC No 204-884-0, registration number: 01-2119490822-33-0000) that is relevant for humans.

Justification for classification or non-classification

No classification in accordance with EU CLP (Regulation (EC) No. 1272/2008). All in vitro tests are negative.