2,6-di-tert-butylphenol

Endpoint summary

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

Reference

Reference 1

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

Guideline:

other: as described in Ames, B.N. et al, Mutation Research 31: pp. 347-364, 1975

GLP compliance:

yes (incl. QA statement)

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^-2mg/plate	5 x 10^-3mg/plate	2.5 x 10^-3mg/plate	5 x 10^-4mg/plate	2.5 x 10^-4mg/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.

Reference 2

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

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Reference 3

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

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

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

Guideline:

other: EEC Directive 87/302, L 133, p. 61-63

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 10^6cells
			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.