6-tert-butyl-2,4-xylenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro Ames Assay - Negative with and without metabolic activation.

In vitro chromosomal aberration assay - Negative with and without metabolic activation.

In vitro Mammilian Cell Gene Mutation Test - By Read Across Negative with and without metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not specified

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: “Test Methods relating to the New Chemical Substances” (Kanpogyo No. 237, Yakuhatsu No. 306, 62Kikyoku No. 303, March 31, 1987).

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

Chromosome aberration

Species / strain / cell type:

mammalian cell line, other: CHL/IU

Details on mammalian cell type (if applicable):

CHL/IU (referred as “CHL”) cell derived from Chinese hamster.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

0 - 0.56 mg/ml

Concentration which clearly exceeded 50% growth inhibition concentration (approximately 60% growth inhibition concentration) was 0.033 mg/mL in direct method, 0.056 mg/mL and 0.029 mg/mL with or without S9 mix in metabolic activation method, respectively. Based on the results, 0.033 mg/mL was selected as high dose for direct method and metabolic activation method without S9 mix, 0.056 mg/mL for metabolic activation method with S9 mix. One half and one quarter of the highest treatment concentration were chosen as the medium and low dose, respectively.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Preparation of culture solution: Eagle MEM culture solution to which 10% fetal bovine serum (FCS, Lot No. 1C2073, JRH BIOSCOENCES) was added was used for incubation. MEM culture solution was prepared by addition of 9.4 g of Eagle MEM medium “Nissui” powder (Nihon Pharmaceutical Co., Ltd.,) to 1 L of distilled water, steam sterilization under pressure for 15 minutes at 121oC followed by addition of 300 mg of L-glutamine (sterilized, Nihon Pharmaceutical Co., Ltd.,) and approximately 12.5 mL of 10% NaHCO3 solution. MEM culture solution of twice of concentration was prepared similarly as the following MEM culture solution by dissolution of 9.4 g of above medium to 500 mL of distilled water.

Culture condition: Two x 104 CHL cells were inoculated to petri dish (6 cm diameter, Corning) containing 5 mL of culture solution and were incubated in CO2 incubator (5% CO2) at 37oC.

Preparation of the test substance: The test substance was freshly prepared before each use. DMSO (Lot No.:12H0658, Sigma Chemical Co.,) was used as solvent. Technical product was dissolved in the solvent (30 mg/mL for growth inhibition test, 6.6 mg/mL and 11.2 mg/mL for chromosome aberration test) to prepare stock solution and the stock solution was serially diluted with the solvent to obtain the test substance preparation at the specified concentrations. The test substance preparation was added to achieve 0.5% (v/v) of the culture medium in all tests. In chromosome aberration test, contents of the test substance solution were measured at high and low dose groups in direct method and metabolic activation method at Analytical Chemistry Laboratory, Hatano Research Institute. As a result of the measurement, concentrations were within acceptable range in all preparations (mean contents in the solvent should be within 90.0 – 110% of added volume).

Test condition: In direct method, cells were incubated for 3 days and then culture medium was discarded. Then 5 mL of culture medium and 25 µL of the test substance solution of each concentration were added to petri dish to conduct 24- and 48-hour continuous treatment.

In metabolic activation test, cells were incubated for 3 days and then culture medium was discarded. Then 3 mL of the mixture which consists of MEM culture medium, MEM culture medium of 2-fold concentration and S9 mix at the ratio of 4:1:1 was added to petri dish. In treatment group without S9 mix, 3 mL of MEM culture medium was added to petri dish, 15 µL of the test substance preparation was added further and then treated for 6 hours. After the completion of treatment, culture medium was freshly replaced and then cultured for further 18 hours. S9 mix was prepared with the following composition:
S9* 3
20 mM HEPES (pH 7.2) 2
50 mM MgCl2 1
330 mM KCl 1
50 mM G-6-P 1
40 mM NADP 1
Distilled Water 1
Total 10 mL
* S9: S9 of Kikkoman Corporation (Lot No: RAA-292, manufactured in May 1993, Lot No.: RAA-297, manufactured in August 1993) which was prepared by treatment of phenobarbital and 5,6-benzoflavone to Sprague-Dawley rat was purchased and stored in deep freezer with -80oC until use.

Cell growth inhibition test: To determine the treatment concentration in chromosome aberration test, effect of the test substance to cell growth was assessed.

Treatment condition: Cell growth inhibition test was conducted with 48 hours treatment group in direct method and treatment groups with or without S9 mix in metabolic activation test. In the preliminary test, clear growth inhibition was observed in a range of 0.11 – 1.8 mg/mL (10 mM) in direct method and metabolic activation method without S9 mix, and in a range above 0.150 mg/mL with S9 mix. Therefore, concentration in a range of 0.005 – 0.150 mg/mL was used in both direct method and metabolic activation method. 2 dishes were used for each concentration.

Specimen preparation: After the completion of incubation, culture medium was discarded and 10% formalin solution was added. Cells were adhered on petri dish and fixed as they were, stained with 0.1% crystal violet solution.

Index for growth inhibition and result: Growth rate of each group was measured using monolayer cultured cells density meter (MonocellaterTM, Olympus Optical Co., Ltd.). The ratio of the growth rate to the solvent control of the test substance treatment group was calculated and the result was used as the index of the growth inhibition effect of the test substance to CHL cell.
As a result, the concentration which clearly exceeded 50% growth inhibition concentration (approximately 60% growth inhibition concentration) in direct method was calculated as 0.033 mg/mL from 2 concentrations which bracketed 60% growth inhibition concentration. The concentrations at which approximately 60% growth inhibition was noted were 0.056 mg/mL and 0.029 mg/mL in metabolic activation method with and without S9 mix, respectively. Although 61% of growth inhibition was noted at 0.15 mg/mL with S9 mix, this was considered to be due to adhesion of the test material to petri dish.

Group scheme in the main test: Based on the result of cell growth inhibition test, 0.033 mg/mL was selected for the high dose groups in direct method and metabolic activation method without S9 mix in chromosome aberration test, 0.056 mg/mL in metabolic activation method with S9 mix.
One half and one quarter concentration of the high dose were selected for the middle and low dose groups, respectively. MC and CPA which were used as positive control materials were prepared by dissolving in water for injection (Lot No.: K2L74, Otsuka Pharmaceutical Factory, Inc.). Each material was used at the concentration which is known to induce chromosome aberration.

Chromosome specimen preparation method
Two hours before completion of incubation, colcemide was added to culture medium to make the final volume of approximately 0.1μg/mL. After the completion of incubation, cells from each group were rinsed with phosphate buffered saline (without CA++ or Mg++), detached by pipetting and collected into centrifuge tube of 10 mL.
The solution was centrifuged for 5 minutes at 1000 – 2000 rpm. After disposal of supernatant, 3 mL of 0.075 M KCl aqueous solution was added to precipitated cells to conduct approximately 30 minutes of hypotonic treatment.
After hypotonic treatment, approximately 6 mL of Carnoy’s flud (glacial acetic acid:methanol = 1:3 v/v) was added to upper phase of hypotonic solution and solution was mixed gently with pipetting from lower phase to fix, and then was centrifuged for 5 minutes at approximately 1000 – 2000 rpm.
After centrifugation, fresh Carnoy’s fluid was added further to the solution to re-suspend cells by pipetting and the solution was centrifuged for 5 minutes at 1000 – 2000 rpm. This operation was repeated for a few times.
To white cell aggregation which was obtained by centrifugation, 0.2 – 0.5 mL of Carnoy’s fluid was added to the solution and it was suspended sufficiently.
Small quantity of cell suspension was dripped onto a slide glass which had been rinsed beforehand and air-dried.
Six slide specimens were prepared for each petri dish respectively.
Test system I.D. number, code number and slide number were noted by pencil on frosting area of slide glass.
Dried slide glass was dyed for approximately 8 minutes with stain solution which was prepared by dilution of 4.5 mL of Giemsa (Merck) by 150 mL of phosphate buffered solution (pH 6.8), was rinsed gently by distilled water and was air-dried.
With order of code number, dyed slide specimen was put in slide case which was identified by test system I.D. number and date of specimen preparation to storage.

Chromosome analysis: Among prepared slide specimens, different slides which were obtained from one petri dish were coded and were observed by more than one observer without knowing each treatment conditions. Observer looked for well spread mitotic figure without dissipation of chromosome. Cells with aberration were noted on data sheet with its location on slide which was expressed by location on stage of microscope.
Analysis of chromosome was conducted according to criteria by Mammalian Mutagenicity Study (MMS) Group of The Japanese Environmental Mutagen Society and was conducted with observation of structural aberration such as chromosome and chromatid gap, breakage and exchange or presence of polyploidy. Generally, 200 mitotic figures for each group were analyzed for structural aberration and 800 mitotic figures for each group for polyploidy.

Evaluation criteria:

For the final judgment for clastogenicity of test substance, below 5% frequency of cells with chromosome aberrations was considered as negative, 5% and above below 10% as pseudo-positive, and above 10% as positive based on the criteria by Ishidate et al.2) When pseudo-positive result was obtained, repeatability or dose dependency was assessed by chromosome aberration test or micronucleus test to implement the final judgment.

Statistics:

Appearance frequency of cells with chromosome aberration was tested for the significance between solvent control and test substance treatment groups and between solvent control and positive control groups by Fisher’s exact probability test method.

Key result

Species / strain:

mammalian cell line, other: CHL/IU

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

24- and 48-hour continuous treatment with TBX showed no induction of structural chromosomal aberration or polyploidy in any treatment group.

Induction of structural chromosomal aberration or polyploidy was not noted in any group treated with TBX for 6 hours excluding high dose group (0.033 mg/mL) with or without S9 mix. The number of metaphase was not enough for evaluation due to cytotoxicity at high dose group.

Cells with structural aberration such as chromatid exchange (cte) or chromatid breakage (ctb) were induced at high frequency in positive control of MC treatment group or CPA treatment group with S9 mix in direct method.

Conclusion
6-tert-butyl-2,4-xylenol did not induce any structural chromosomal aberrations or polyploidy in any treatment group at any concentration including the concentration at which clearly exceeded 50% growth inhibition concentration after 24- and 48-hour continuous treatment in direct method (0.008 – 0.033 mg/mL).

Also in metabolic activation method, the test substance did not induce any structural chromosomal aberrations or polyploidy in any treatment group at any concentration including the concentration at which clearly exceeded 50% growth inhibition concentration after 6-hour treatment with S9 mix (0.014 – 0.028 mg/mL) or without S9 mix (0.008 – 0.017 mg/mL).

Therefore, it is concluded that 6-tert-butyl-2,4-xylenol does not induce chromosomal aberrations to in vitro CHO cells under above stated test condition.

Note: No unexpected events which may have adversely affected the reliability of the test or no deviation from the test protocol were noted during the study.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1 Growth inhibition of CHL cells continuously treated with 6-tert-butyl-2,4-xylenol (TBX) for 48 hours without S9 mix

Concentration of TBX (mg/ml)	Cell growth (% of control)
			Average
0	100	100	100.0
0.005	100	96	98.0
0.009	93	92	92.5
0.019	83	87	85.0
0.038	27	27	27.0
0.075	0	0	0.0
0.150	0	0	0.0

Cell growth was measured by Monocellater^TM(OLYMPUS)

 

Table 2 Growth inhibition of CHL cells treated with 6-tert-butyl-2,4-xylenol (TBX) for 6 hours withS9 mix

Concentration of TBX (mg/ml)	Cell growth (% of control)
			Average
0	100	100	100.0
0.005	104	108	106.0
0.009	93	99	96.0
0.019	82	88	85.0
0.038	60	73	66.5
0.075	16	12	14.0
0.150	48	74	61.0

Cell growth was measured by Monocellater^TM(OLYMPUS)

 

Table 3 Growth inhibition of CHL cells treated with 6-tert-butyl-2,4-xylenol (TBX) for 6 hours without S9 mix

Concentration of TBX (mg/ml)	Cell growth (% of control)
			Average
0	100	100	100.0
0.005	100	107	103.5
0.009	94	96	95.0
0.019	83	92	87.5
0.038	0	0	0.0
0.075	0	0	0.0
0.150	0	0	0.0

Cell growth was measured by Monocellater^TM(OLYMPUS)

 

Table 4 Chromosome analysis of Chinese hamster cells (CHL) continuously treated with 6-tert-butyl-2,4-xylenol (TBX)** without S9 mix

Group	Concentration (mg/ml)	Time of exposure (hr)	No. of cells analysed	No. of structural aberrations								Others^3)	No. of cells with aberrations				Polyploid^4)(%)	Judgement^5)
				gap	ctb	cte	csb	cse	f	mul ^2)	total		TAG (%)		TA (%)			SA	NA
Control			200	0	0	0	0	0	0	0	0	0	0	(0.0)	0	(0.0)	0.38
Solvent^1)	0	24	200	0	0	1	1	0	0	0	2	0	2	(1.0)	2	(1.0)	0.13
TBX	0.008	24	200	0	0	0	1	0	0	0	1	0	1	(0.5)	1	(0.5)	0.50	-	-
TBX	0.017	24	200	0	0	0	0	1	0	0	1	0	1	(0.5)	1	(0.5)	0.50	-	-
TBX	0.033	24	200	2	2	4	1	0	1	0	10	0	7	(3.5)	6	(3.0)	0.00	-	-
MC	0.00005	24	200	4	74	127	4	0	3	10	223	4	106*	(53.0)	105*	(52.5)	0.00	+	-
Solvent^1)	0	48	200	1	0	0	0	0	0	0	1	0	1	(0.5)	0	(0.0)	0.13
TBX	0.008	48	200	0	0	0	0	0	0	0	0	0	0	(0.0)	0	(0.0)	0.00	-	-
TBX	0.017	48	200	0	0	0	2	0	1	0	3	0	2	(1.0)	2	(1.0)	0.13	-	-
TBX	0.033	48	200	0	3	1	0	1	1	0	6	0	3	(1.5)	3	(1.5)	0.25	-	-
MC	0.00005	48	200	7	49	152	5	8	10	30	261	16	113*	(56.5)	112*	(56.0)	0.50	+	-

Abbreviations: gap: chromatid gap and chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring etc.), f: acentric fragment (chromatid type), mul: multiple aberrations, TAG: total no. of cells with aberrations, TA: total no. of cells with aberrations except gap, SA: structural aberration, NA: numerical aberration, MC: mitomycin C. 1) Dimethyl sulfoxide was used as solvent. 2) More than ten aberrations in a cell were scored as 10. 3) Others, such as attenuation and premature chromosome condensation, were excluded from the no. of structural aberrations. 4) Eight hundred cells were analysed in each group. 5) Judgement was done on the basis of the criteria of Ishidate et al. (1987). *: Significantly different from solvent control at p<0.05. **: Purity was 98.5%.

 

Table 5 Chromosome analysis of Chinese hamster cells (CHL) treated with 6-tert-butyl-2,4-xylenol (TBX)** with and without S9 mix

Group	Concentration (mg/ml)	S9 mix	Time of exposure (hr)	No. of cells analysed	No. of structural aberrations								Others^3)	No. of cells with aberrations				Polyploid^4)(%)	Judgement^5)
					gap	ctb	cte	csb	cse	f	mul ^2)	total		TAG (%)		TA (%)			SA	NA
Control				200	1	3	0	0	0	0	0	4	1	3	(1.5)	2	(1.0)	0.13
Solvent^1)	0	-	6-(18)	200	0	0	0	2	0	0	0	2	0	1	(0.5)	1	(0.5)	0.00
TBX	0.008	-	6-(18)	200	1	0	0	2	0	0	10	13	0	3	(1.5)	2	(1.0)	0.38	-	-
TBX	0.017	-	6-(18)	200	0	0	1	2	0	1	0	4	0	3	(1.5)	3	(1.5)	0.13	-	-
TBX	0.033	-	6-(18)	1	0	0	0	0	0	0	0	0	0	0	(0.0)	0	(0.0)	0.00^6)	Tox	Tox
CPA	0.005	-	6-(18)	200	0	0	1	0	0	1	0	2	0	2	(1.0)	2	(1.0)	0.75*	-	-
Solvent^1)	0	+	6-(18)	200	2	3	0	0	0	0	0	5	1	4	(2.0)	2	(1.0)	0.63
TBX	0.014	+	6-(18)	200	0	1	0	1	0	1	0	3	0	3	(1.5)	3	(1.5)	0.13	-	-
TBX	0.028	+	6-(18)	200	1	1	1	1	0	1	0	5	1	3	(1.5)	2	(1.0)	0.38	-	-
TBX	0.056	+	6-(18)	10	0	6	13	0	1	1	0	21	0	8*	(80.0)	8*	(80.0)	0.00^7)	Tox	Tox
CPA	0.005	+	6-(18)	200	15	126	320	12	3	2	100	578	3	157*	(78.5)	157*	(78.5)	0.25	+	-

Abbreviations: gap: chromatid gap and chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring etc.), f: acentric fragment (chromatid type), mul: multiple aberrations, TAG: total no. of cells with aberrations, TA: total no. of cells with aberrations except gap, SA: structural aberration, NA: numerical aberration, CPA: cyclophosphamide. 1) Dimethyl sulfoxide was used as solvent. 2) More than ten aberrations in a cell were scored as 10. 3) Others, such as attenuation and premature chromosome condensation, were excluded from the no. of structural aberrations. 4) Eight hundred cells were analysed in each group. 5) Judgement was done on the basis of the criteria of Ishidate et al. (1987). 6) One cell was analysed. 7) Eleven cells were analysed. *: Significantly different from solvent control at p<0.05. **: Purity was 98.5%.

Conclusions:

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

6-tert-butyl-2,4-xylenol does not induce chromosomal aberrations to in vitro CHO cells under above stated test condition.

Executive summary:

As a part of the investigation project of toxicity regarding the safety check for existing chemicals by OECD, in vitro chromosome aberration test withcultured Chinese hamster cells (CHL/IU) was conducted to assess cytogenetic effect of 6-tert-butyl-2,4-xylenol on cultured cells.

 

Above test was conducted in compliance with “Test Methods relating to the New Chemical Substances” (Kanpogyo No. 237, Yakuhatsu No. 306, 62Kikyoku No. 303, March 31, 1987) and “OECD Guidelines for Testing of Chemicals: 473” and based on “GLP Standards for Chemical Substance” (Kanpogyo No. 39, Yakuhatsu No. 229, 59 Kikyoku No. 85, March 31, 1984; revised by Kankiken No. 233, Eisei No. 38, 63 Kikyoku No. 823, November 18, 1988).

 

Concentration which clearly exceeded 50% growth inhibition concentration (approximately 60% growth inhibition concentration) was 0.033 mg/mL in direct method, 0.056 mg/mL and 0.029 mg/mL with or without S9 mix in metabolic activation method, respectively. Based on the results, 0.033 mg/mL was selected as high dose for direct method and metabolic activation method without S9 mix, 0.056 mg/mL for metabolic activation method with S9 mix. One half and one quarter of the highest treatment concentration were chosen as the medium and low dose, respectively. 24- and 48-hour continuous treatment was performed in direct method, 6-hour treatment was performed in metabolic activation method with or without S9 mix (with 18-hour recovery period) and specimen was prepared. These were subjected to microscopy to assess the clastogenicity.

 

Induction of structural chromosomal aberration or polyploid cell was not observed in any treatment group of CHL/IU cells either in 24-hour or 48-hour continuous treatment in direct method. In metabolic activation method, analysis of enough number of cells was not possible due to cytotoxicity at high dose groups in 6-hour treatment with or without S9 mix (0.056 mg/mL and 0.033 mg/mL, respectively). However, induction of structural chromosomal aberration or polyploid cell was not observed in other treatment groups.

 

Clastogenicity of 6-tert-butyl-2,4-xylenol was assessed with cultured Chinese hamster cells (CHL/IU) and the result was negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Presence or absence of mutagenicity of 6-tert-butyl-2,4-xylenol was initially examined by reverse mutation test on bacteria.  Range-finding test was performed by plate method under the condition of direct method and metabolic activation method using 5 strains of Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA as the tester bacteria in the dose range of 50 to 5000 µg/plate. As a result, anti-bacterial effect was observed for all tester bacteria. Therefore, the definitive test was performed in the dose range of 6.25 to 200µg/plate under condition of direct method, and in the dose range of 6.25 to 200 µg/plate for Salmonella typhimurium TA1537 and in the dose range of 12.5 to 400 µg/plate for other 4 tester bacteria under the condition of metabolic activation method.

 

As a result, over 2 folds increase of revertant colony number compared to negative control group was not observed for 5 tester bacteria at any dose level of the test material in the two definitive tests. Therefore, it was judged that 6-tert-butyl-2,4-xylenol had no mutagenicity (negative) in the test system used.

 

An in vitro chromosome aberration test withcultured Chinese hamster cells (CHL/IU) was conducted to assess cytogenetic effect of 6-tert-butyl-2,4-xylenol on cultured cells.

 

Induction of structural chromosomal aberration or polyploid cell was not observed in any treatment group of CHL/IU cells either in 24-hour or 48-hour continuous treatment in direct method. In metabolic activation method, analysis of enough number of cells was not possible due to cytotoxicity at high dose groups in 6-hour treatment with or without S9 mix (0.056 mg/mL and 0.033 mg/mL, respectively). However, induction of structural chromosomal aberration or polyploid cell was not observed in other treatment groups. 6-tert-butyl-2,4-xylenol was not considered to be clastogenic to the cultured Chinese hamster cells (CHL/IU) utilised in the test.

 

Evaluation of the mutagenic activity in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells was conducted utilising read across to the structural analogue, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol. The substance, CAS85-60-9, is analogous to the substance to be registered, but is approximately twice the size of CAS 1879-09-0 – this is essentially a second “duplicate” molecule of the substance to be registered. Given the results of the mutagenicity tests on the substance itself (negative) along with the fact that the substance is not known to cause mutagenic effects based on many years experience of use, it was deemed appropriate to assess the final required mutagenicity endpoint by read across. CAS 85-60-9 is likely to pose more of a risk of mutagenicity, as there are twice the number of substituted phenol groups available to case effects. It is acknowledged that the difference in molecular size may impact on the results, but it is deemed that the additional substituted phenol group present inCAS85-60-9 would outweight any likely steric effects, if the substance was truly mutagenic.

 

The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

 

In the first experiment, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested up to concentrations of 15 and 50 μg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested up to cytotoxic levels of 74 and 46% in the absence and presence of S9-mix, respectively. 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol precipitated in the culture medium at dose levels of 35 μg/ml and above.

 

In the second experiment, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was tested up to concentrations of 17.5 and 70 μg/ml in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours in the absence of S9-mix and 3 hours for incubations in the presence of S9-mix. The test substance tested up to cytotoxic levels of 94 and 83% in the absence and presence of S9-mix, respectively. 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol precipitated in the culture medium at dose levels of 50 μg/ml and above.

 

In the absence of S9-mix, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

 

In the presence of S9-mix, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

 

It is concluded that 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

Justification for selection of genetic toxicity endpoint

Study performed in accordance with OECD guideline no. 473. 3 studies are available, all negative.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP and in accordance with appropriate OECD Guideline. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability.

 

The above results triggered no classification under the CLP Regulation (EC No 1272/2008). No classification is therefore required.