2,6-xylidine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

2,6-DMA did not induce mutations in various tests with Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 and TA1538 in the presence or absence of metabolic activation. However, a weak positive response was observed with TA100 in the presence of metabolic activation.

2,6-DMA induced mutations in mouse L5178Y lymphoma cells.

The substance also induced sister chromatid exchange and chromosomal aberrations at cytotoxic concentrations in Chinese hamster ovary cells with and without exogenous metabolic activation.

Link to relevant study records

Reference

Reference 1

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Principles of method if other than guideline:

A chromosome aberration test was performed with CHO cells for 108 test chemicals.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2,6-xylidine
- no further data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

WB1

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction (Aroclor induced)

Test concentrations with justification for top dose:

900-1400 µg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

mitomycin C

Evaluation criteria:

For chromosome aberrations, linear regression analysis of the a single percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, and the test was that described by Margolin et al [1983, pp 714-715].The P values were adjusted by Dunnett's method to take into account the multiple dose comparisons . For data analysis, we used the "total" aberration category, and the criterion for a positive response was that the adjusted P value be <0.05.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The aberration tests with and without S9 were positive rather at toxic doses and in the presence of precipitate.
Cytotoxic effects at 900 µg/ml, precipitates; significant at 1000 µg/ml (- S9) or 1200 µg/ml (+ S9).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four strains tested; no strain included to identify cross-linking mutagens

Principles of method if other than guideline:

Standard plate incorporation according to Ames et al. (1975). Results of 300 tested chemicals were listed in tabular form.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2,6-Xylidine
- Supplier: Ethyl Corporation,
- Analytical purity: 99.1 % (Analyzed by Midwest Research Institute, Kansas City, MO .)
- no further data.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

rat and hamster liver S9 mix (Aroclor-induced)

Test concentrations with justification for top dose:

10-3333 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-henylenediamine: -S9 (TA98), 2-aminoanthracene: +S9 (all strains)

Details on test system and experimental conditions:

Tester strains
Salmonella typhimurium strains TA97, TA98, TA100, TA1535, and TA1537 were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831 . Cultures were grown overnight with shaking at 37° C in Oxoid No.2 broth, and their phenotypes were analyzed prior to their use for mutagenicity assays.

Preparation of Liver S-9 Fractions
The S9 (9,000g supernatant) fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared as described previously [Haworth et al, 1983]. The S9 mixes were prepared immediately prior to use and contained either 10 % or 30 % S9; occasionally, other levels were used. All chemicals were tested in the absence of
metabolic activation and with rat and hamster S-9 fractions.

Test Protocol
The preincubation assay was performed as described previously [Haworth et. al., 1983], with some differences, as described below. The test chemical (0.05 ml), Salmonella culture (0.1 ml), and S-9 mix or buffer (0.5 ml) were incubated at 37°C, without shaking, for 20 min. Chemicals known or suspected to be volatile were incubated in capped tubes. The top agar was added and the contents of the tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium [Vogel and Bonner, 1956]. The histidine-independent (his+) colonies arising on these plates were counted following two days incubation at 37°C. Plates were
machine counted (New Brunswick, Edison, NJ; Artek, Farmingdale, NY) unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the background agar. At the discretion of the investigators, plates with low numbers of colonies were counted by hand. Variations in the protocol among the tested chemicals reflect the evolution of the protocol originally described by Haworth et al. [1983] . Four protocol variations are evident from the data in Appendix 2 .
1) Testing in strains TA97, TA98, TA100, and TA1535, with some additional testing in strain TA1537 ; 10% S-9 was used.
2) The first test of a chemical was without activation and with 10 % S-9 in the S-9 mix. If a positive result was obtained the test was repeated. If the tests were negative they were repeated without S-9 and with 30 % S-9.
3) The order of use of 10% and 30% S9 was reversed.
4) Initial testing was in strains TA98 and TA 100 without activation and with 30 % rat and hamster S9s. If a positive result was obtained in one of these two strains it was repeated and the other strains were not used. If the tests were negative, the other strains were used with 30 % and10 % S9. A chemical was not designated non-mutagenic unless it had been tested in strains TA98, TA100, TA1535, and TA97 and/or TA 1537, without activation and with 10 % and 30 % rat and hamster S9. Occasionally, 5 % S9 was also used in all protocol variations. All chemicals were tested initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay . The toxicity assay was performed using TA 100 or the system developed by Waleh et al . [1982]. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both. Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in the preliminary toxicity test. Subsequent trials occasionally used narrower dose increments and may not have included doses in the toxic range. Chemicals that were not toxic were tested, with few exceptions, to a maximum dose of 10 mg/plate. Chemicals that were poorly soluble were tested up to doses defined by their solubilities. At least five doses of each chemical were tested in triplicate. Experiments were repeated at least one week following the initial trial. A maximum of 0.05 ml solvent was added to each plate. Concurrent solvent and positive controls were run with each trial. The positive controls in the absence of metabolic activation were sodium azide (TA1535 and TA100), 9-aminoacridine (TA97 and TA1537), and 4-nitro-o-henylenediamine
(TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.
Occasionally, a laboratory used a single solvent and/or positive control for more than one chemical tested on the same day. Each laboratory made its own determination regarding positive control dose levels.

Evaluation criteria:

The data were evaluated as described previously [Zeiger et al. 1987]. Evaluations were made at both the individual trial and overall chemical levels. Individual trials were judged mutagenic (+), weakly mutagenic (+W), questionable (?), or non-mutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of "+W," if only a single dose was elevated over the control, or if the increase seen was not dose related. The distinctions between a questionable mutagenic response and a non-mutagenic or weak mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach twofold over background for a chemical to be judged mutagenic. A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible dose-related response over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a "+W" response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged non-mutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or non-mutagenicity.

Species / strain:

other: Salmonella typhimurium; TA 97, 98, 100, 1535, 1537

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

2,6-xylidine produced only weak positve results with the tester strains investigated. Results of 3 independent laboratories judged the test substance as weakly positive ( Microbiological Associates, Inc . (MIC) and SRI International (SRI)) or negative (Case Western Reserve University (CWR) ).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Qualifier:

no guideline followed

Principles of method if other than guideline:

no details indicated

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2,6-Xylidine
- no further data.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

not indicated

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

Mouse lymphoma assay

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

L5178Y mouse lymphoma cell colonies selected in trifluorothymidine have a bimodal size distribution in response to some chemicals, with the larger colonies thought to result from point mutations, and the smaller colonies from larger chromosomal defects. However, the relative induction of the two types of colonies has not been measured in previous mutagenicity screening programs. For this study, 21 coded chemicals were tested, with and without activation (S-9); the total number of colonies was counted using an Artek Model 880 counter and the distributions of colony sizes were determined where the given chemical increased the overall mutation frequency. 2,6-xylidine was positive both with and without S-9; In most of the positive experiments more small colonies than large colonies were induced. This may indicate that these chemicals are clastogenic as well as mutagenic.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Feeding or injection of 2,6-DMA had no mutagenic effect in a sex-linked recessive lethal assay in Drosophila melanogaster.

No increase of micronuclei was detected in the bone marrow of male mice after oral doses of up to 375 mg/kg.

2,6-DMA did not induce unscheduled DNA synthesis in rat hepatocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)

Principles of method if other than guideline:

Seventy chemicals were tested for the ability to induce sex-linked recessive lethal (SLRL) mutations in postmeiotic and meiotic germ cells of male Drosophila melanogaster.

GLP compliance:

not specified

Type of assay:

Drosophila SLRL assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Xylidine
- Supplier: Ethyl Corporation,
- purity: 99.1 %
- Batch No.: E121279/01,
- no further data

Species:

Drosophila melanogaster

Strain:

other: Canton-S and Basc

Sex:

male

Route of administration:

other: feeding and injection

Duration of treatment / exposure:

3 x 2 - 3 days or single injection

Dose / conc.:

100 other: mg/L (feeding)

Dose / conc.:

4 000 other: mg/L (injection)

Control animals:

yes, concurrent vehicle

Positive control(s):

A total number of 70 chemicals were included in this study among them a number of test chemicals considered positive in the experiment including 4,4'-methylenedianiline 2HCI, 3-(chloromethyl) pyridine HCI, HC blue 1, maloxon, cis-dichlorodiaminoplatinum, 1,2-pro-dibromoethane, dibromomannitol, 1,2-epoxypropane, glycidol, myleran, toluene diisocyanate.
.

Statistics:

For the SLRL assay, a minimum of about 5,000 chromosomes was scored from each of the treated and concurrent control groups unless the mutant frequency exceeded 1 %. If two or more lethals were recovered among the progeny of one male, a Poisson analysis [Owen, 19621 was performed to determine if these were part of a "cluster ." (A cluster is defined as a group of mutated sperm cells derived from a single mutational event occurring in a spermatogonial cell.) Clusters must be spontaneous in origin, because only meiotic and postmeiotic stages of spermatogenesis were treated. Therefore in those cases in which a male was determined to have produced a cluster the lethal and nonlethal tests for that P1 male were removed from the data. The corrected treated and control data were compared using a normal approximation to the binomial distribution, as suggested by Margolin et al. [1983]. In addition, the treated data were compared to the historical control as described by Mason et al. [1992]. For a compound to be considered mutagenic, the mutant frequency in the treated series (treated frequency must exceed 0.15 % with a P value of < 0.05, or the treated frequency exceed 0.10 % with a P value of < 0.01. If the treated frequency is between 0.10 % and 0.15 % and the P value is between 0.1 and 0.01, or if the treated frequency is higher than 0.15 % and the P value is between 0.1 and 0.05, the result is considered equivocal. All other results are considered negative. Translocation data for each treated sample were compared to the historical control data for that laboratory using a conditional binomial test [Kastenbaum and Bowman, 1970]. As a rule, at least two translocations required among about 5,000 tests in the treated series for a compound considered positive. As a comparison, the combined historical translocation rate for three laboratories, including 33.783 new tests is 4/149,946 (0.0027%).

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Table 1: Summary of results

Dose of 2,6-Xylidine	ROA	% mortality	% sterility	Lethals			Tests			Total lethals	Total tests	% lethals
				BR1	BR2	BR3	BR1	BR2	BR3
100	feeding	5	0	0	2	6	2,211	1,942	1,821	8	5,974	0,13
0				4	2	10	1,934	2,128	2,046	16	6,108	0,26
4000	injection	14	0	4	1	3	1,911	1,793	1,669	8	5,373	0,15
0				0	0	6	1,999	1,821	1,811	6	5,631	0,11

One cluster of 4 lethals in treated injection experiment .

Conclusions:

Negative: no increase in the frequency of sex-linked recessive lethals was found.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

not specified

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2,6-Xylidine
- Supplier: Radian Corporation
- no further data

Species:

rat

Strain:

Fischer 344

Sex:

male

Route of administration:

oral: gavage

Duration of treatment / exposure:

single administration

Dose / conc.:

40 mg/kg bw (total dose)

Dose / conc.:

200 mg/kg bw (total dose)

Dose / conc.:

850 mg/kg bw (total dose)

No. of animals per sex per dose:

3 animals per dose

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

Cell Culture, Fixation, and Staining
Cultures were incubated in WE containing 10 μCi/ml 3H-(methyl)-thymidine (specific activity, approximately 80 Ci/mmol) for 4 hr at 37°C, 5% CO,, followed by 14-18 hr in WE containing 0.25 mM unlabeled thymidine. The cultures were then washed twice with WE, followed by 10 min in 1% sodium citrate to swell cells, fixed in 1 :3 glacial acetic acid :ethanol for 30 min, and washed 3 to 6 times with deionized water. The dried coverslips were mounted to glass slides using Paramount. The slides were dipped in Kodak NTB-2 nuclear track emulsion diluted 1 : 1 with deionized water, and exposed at -20°C for 7-14 days and then developed and stained as previously described [Mitchell and Mirsalis, 1984].

Evaluation criteria:

UDS measurement:
Compounds were considered negative if the NG of all dose groups was a negative number and the %IR was less than 10%. Compounds were considered positive if the average NG of any dose group exceeded 0 NG.

Statistics:

no data

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Table. 1: Summary of results

Compound	Dose (mg/kg)	Time (hr)	Male rat
			NG	(n)	% IR
Corn oil	-	2	-6.4 ± 2.9	2	1 ± 0
	-	12	-5.6 ± 0.4	52	2 ± 0
Control/water	-	2	-5.6 ± 0.5	31	2 ± 0
2-Acetaminofluorene	50	12	22.9 ± 1.7	52	85 ± 2
2,6-xylidine	40	2	-5.9 ± 1.2	3	4 ± 1
		12	-7.0 ± 1.4	3	4 ± 3
	200	2	-7.1 ± 0.7	3	2 ± 0
		12	-6.9 ± 0.3	3	1 ± 0
	850	2	-6.7 – 1.0	3	1 ± 1
		12	-10.3 ± 0.5	3	0

Conclusions:

2,6-xylidine failed to induce UDS in rats

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

SCOEL (2009):

 

2,6-DMA is a carcinogen in rats causing invasive tumours of the nasal cavity. These tumours develop in tissues which show inflammatory processes at the same time and prior to the occurrence of tumours. It remains open whether inflammatory and toxic effects play a rote in tumour development. However, 2,6-DMA is N-oxidised to genotoxic products, binds to DNA at the target tissue, and forms a type of DNA-adduct analogue to that observed with genotoxic carcinogenic polycyclic arylamines. Hence, the mode of action of nasal tumour development in experimental animals remains open. Under these conditions, no health-based OEL can be derived. Moreover, no inhalation study exists to derive quantitative risk estimates.

Additional information

SCOEL (2009):

 

In vitro

2,6-DMA or its hydrochloride did not induce mutations in various tests with Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 and TA1538 in the presence or absence of exogenous metabolic activation system. However, a weak positive response was observed in preincubation and plate incorporation tests with TA100 in the presence of metabolic activation.

The substance was not active in a DNA damage and repair assay with E. coli. K-12 343/113, uvrB-/recA- and uvrB+/recA+.

2,6-DMA induced mutations in mouse L5178Y lymphoma cells.

The substance also induced sister chromatid exchange and chromosomal aberrations at cytotoxic concentrations in Chinese hamster ovary cells with and without exogenous metabolic activation (ECB, 2000; NLM, 2006).

A weakly positive effect was also observed in a cell transformation assay with Balb/c 3T3 cells (ECB, 2000).

 

N-Hydroxy-2,6-DMA, the product of metabolic activation of 2,6-DMA in vivo, is mutagenic in S. typhimurium TA100 (Jeffrey et al., 2002). In a series of N-(hydroxymethyl)-, N-(hydroxydimethyl)- and N-hydroxyethyl)-aniline derivatives, N-hydroxy-2,6-DMA was the most mutagenic compound when tested in S. typhimurium TA100 in the absence of metabolic activation (Nohmi et al., 1984; Marques et al., 1997).

 

The products of N-oxidation of 2,6-DMA form DNA adducts. Different DNA adducts resulting from the reaction of N-acetoxy-2,6-DMA were identified. Three of the adducts were reaction products of the exocyclic heteroatoms of deoxyadenosine and deoxyguanosine with the carbon atom in para position to the arylamine nitrogen. However, the fourth adduct resulted from reaction of the 2,6-DMA nitrogen with the C8 atom of deoxyguanosine (Marques et al., 2002). Such N-(desoxyguanosine-8-yl)- adducts are the same as those which are found in case of carcinogenic polycyclic aryl amines (Greim, 1998). However, in contrast to the usual pattern obtained with aromatic amines, where C-8-substituted deoxyguanosine adducts predominate, the adduct profile for N-acetoxy-2,6-DMA was different as the other three adducts mentioned above were obtained with higher yields (Goncalves et al., 2001).

 

In vivo

No genotoxic effect an E. coli K12 was observed in a host-mediated assay after feeding of 2,6-DMA to mice (ECB, 2000).

Feeding or injection of 2,6-DMA had no mutagenic effect in a sex-Iinked recessive lethal assay in Drosophila melanogaster (ECB, 2000).

No increase of micronuclei was detected in the bone marrow of male mice after oral doses of up to 375 mg/kg d (Parton et al, 1988).

2,6-DMA did not induce unscheduled DNA synthesis in rat hepatocytes (Mirsalis et al., 1989).

However, covalent binding of 2,6-DMA to DNA was observed in the nasal epithelium of rats after gavage administration of 2,6-DMA (Duan et al., 2004).

In another study, binding in liver and ethmoid turbinates of rats were observed after oral treatment with 2,6-DMA, and the covalent binding index increased after 9 days of pre-treatment with 2,6-DMA (Short et al., 1989b). In this and in a further study (Jeffrey et al., 2002), the level of covalent binding was higher in ethmoid turbinates than in liver tissue.

Conclusion:

With respect to genotoxicity, there are negative (Ames with rat and also hamster S-9 mix which is more effective in activation of aromatic amines, DNA damage and repair) and positive (SCE, chromosome aberration, mouse lymphoma) in vitro tests, yet all in vivo tests (SLRL in Drosophila, micronucleus test, UDS test, host-mediated assay) were negative. However, no toxicity was seen in the in vivo MNT and therefore there is no proof that the test substance reached the bone marrow after oral application. DNA binding in pretreated rats (9 days with 262.5 mg/kg/d cold material) after i.p. administration was found in liver (slight, CBI 7 .9) and nasal turbinates (moderate, CBI 41.9), whereas a single application was negative; thus, only after massive repeated application of the test substance DNA binding occurred. On the basis of the current data, the classification criteria with respect to mutagenicity are not met. However, the mutagenic potential of the test substance remains unclear until further studies are available.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2018/1480.