Butyraldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a reverse gene mutation assay in bacteria (conducted according to OECD TG 471) , strains TA1535, TA1537, TA98 and TA100 of S. typhimurium and E.coli WP2 uvrA pKM101 were exposed to Butyraldehyde in water at concentrations of 5, 16, 50, 160, 500, 1600 and 5000 µg/plate in the presence and absence of mammalian metabolic activation.

Butyraldehyde was tested up to limit concentration of 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable and it satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-09-20 to 2022-10-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD 1997, as corrected in 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of the test material used in the study report: Butyraldehyde
- Appearance: Colourless liquid
- Storage condition of test material: 15°C to 25°C, protected from light. Store under nitrogen.
- Stability under test conditions: no stability analyses undertaken, as fresh preparations of the test article was employed

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: The E. Coli strain was specifically WP2 uvrA pKM101

Metabolic activation:

with and without

Metabolic activation system:

S-9 Fraction obtained from the livers of male Sprague Dawley rats induced with β-Naphthoflavone/Phenobarbital.

Test concentrations with justification for top dose:

Final concentrations: 5, 16, 50, 160, 500, 1600, 5000 μg/plate

Vehicle / solvent:

Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

5.0 µg/plate; positive control for strain TA100 and TA1535 in the presence of S9-mix. 10.0 µg/plate; positive control for strain WP2 uvrA pKM101 in the presence of S9-mix.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

5.0 µg/plate; positive control for strain TA98 and TA1537 in the presence of S9-mix.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

2.0 µg/plate; positive control for strain WP2 uvrA pKM101

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate; positive control for strain TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2.0 µg/plate; positive control for strains TA1535 and TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

2.0 µg/plate; positive control for strain TA98

Details on test system and experimental conditions:

Two independent mutagenicity assays were performed, using triplicate plates without and with S-9 for test article, vehicle and positive controls. A preliminary toxicity test was not conducted, because excessive toxicity was not expected. Therefore, the toxicity test was incorporated into the first mutagenicity assay. The following sequence of additions were supplemented to molten agar plates: 0.1 mL of bacterial culture, 0.1 ml of test article solution/vehicle control/positive control and 0.5 mL of 10% S-9 mix or buffer solution. The ingredients were rapidly mixed and the mix was immediately poured onto agar plates. The plates were inverted and incubated at 34 to 39°C for 3 days. Revertant colonies were then counted electronically using automated colony counter. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and sodium phosphate buffer.

Rationale for test conditions:

Standardized test conditions: highest concentration not limited by toxicity or solubility.

Experiment 2 included an incubation in air-tight sealed containers for 3 days following treatment. This was to limit any oxidation of the test article.

Evaluation criteria:

The test article was considered to have provided a mutagenic response if the assay data were valid, and:

1. Treatments with the test article provided a concentration-related increase in revertant numbers at one or more concentrations in at least one strain with or without metabolic activation system

2. An increase in mean revertant colony numbers per plate was observed which was ≥2-fold (in strains TA98, TA100, WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values

3. Any increase in revertant numbers was reproducible, where applicable

The test article was considered positive in this assay if all of the above criteria were met.

The test article was considered negative in this assay if not all the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

No statistical analysis were performed; not required for this study type.

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The substance was not toxic to the bacteria (evidenced by the absence of a drastic decrease in the mean number of revertant colonies). In both the absence and presence of S9 mix in all strains tested, the test substance did not cause a reproducible two-fold or greater increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the vehicle, and did not give evidence of a dose response. The positive controls gave the expected increase in the mean number of his+ revertants both with and without S9. From the data it can be seen that vehicle control counts fell within or close to the laboratory’s historical ranges. The study demonstrated correct strain and assay functioning and was accepted as valid. Five analysable concentrations were obtained in each experiment.

It was concluded that n-Butyraldehyde is not mutagenic up to concentrations of 5000 µg/plate.

Conclusions:

It was concluded that the results obtained with the test substance n-Butyraldehyde in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 as well as E. coli WPS uvrA pKM101 in both the absence and in the presence of the S9-mix indicate that n-Butryaldehyde was not mutagenic under the conditions employed in this study.

Executive summary:

In a reverse gene mutation assay in bacteria (conducted according to OECD TG 471) , strains TA1535, TA1537, TA98 and TA100 of S. typhimurium and E.coli WP2 uvrA pKM101 were exposed to Butyraldehyde in water at concentrations of 5, 16, 50, 160, 500, 1600 and 5000 µg/plate in the presence and absence of mammalian metabolic activation.

Butyraldehyde was tested up to limit concentration of 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable and it satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In a study conducted by Witt et al (2000) the mouse peripheral blood micronucleus (MN) test was performed on samples collected from a sub-chronic toxicity study conducted by the National Toxicology Program (NTP). The incidence on MN polychromatic erythrocytes (PCE) provided an index of damage induced within 72 hours of sampling whereas the incidence of MN in the normocromatic erythrocytes (NCE) population at steady state provided an index of the average damage during the 30 -day period preceding sampling.

In a B6C3F1 mouse peripheral blood micronucleus assay, mice were treated by oral gavage with Butyraldehyde at doses of 0, 0.075, 0.15, 0.3, 0.6 and 1.2  g/kg bw. The vehicle was corn oil. Blood samples were taken at the day of termination. There was not a significant increase in the frequency of micronucleated NCE and PCE in the mouse peripheral blood micronucleus test.

 

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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:

other: Secondary literature source

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mouse peripheral blood micronucleus test was performed on samples collected from a 90 day subchronic toxicity study.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

Mice provide a convenient model for the study because unlike rats, mice do not selectively remove micronucleated cells from the peripheral circulation. Therefore, the frequency of micronucleated cells in the circulating blood of mice would be expected to closely reflect the frequency in bone marrow, and unlike bone marrow sampling, repeated peripheral blood measurements can be made, without disturbing the physiology of the animals, by sampling a few microliters of blood at a time.

Sex:

male/female

Details on test animals or test system and environmental conditions:

No information provided

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

No information provided

Duration of treatment / exposure:

90 days

Frequency of treatment:

No information provided

Post exposure period:

No information provided

Dose / conc.:

0.075 other: g/kg

Dose / conc.:

0.15 other: g/kg

Dose / conc.:

0.3 other: g/kg

Dose / conc.:

0.6 other: g/kg

Dose / conc.:

1.2 other: g/kg

No. of animals per sex per dose:

Number o animals that were analysed in the end of the study:

Males
Control: 10
0.075 g/kg: 9
0.15 g/kg: 10
0.3 g/kg: 10
0.6 g/kg: 10
1.2 g/kg: 5

Females
Control: 10
0.075 g/kg: 9
0.15 g/kg: 7
0.3 g/kg: 9
0.6 g/kg: 10
1.2 g/kg: 4

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control group was not included in the test. This is because the toxicity bioassay from which the test animals were obtained did not include a positive control group. However, positive control slides were included in the experiment to control for staining and scoring procedures.

Tissues and cell types examined:

Erythrocytes

Details of tissue and slide preparation:

Blood was obtained immediately before or at the time of sacrifice (90 days).

Drops of blood were spread on precleaned standard glass microscope slides, air dried, and fixed in absolute methanol for 5 min. All slides were coded prior to scoring by a person not involved in reading the slides.

Evaluation criteria:

Identification of micronucleus (MN) was based on fluorescence emission characteristic of the fluorescent stain used (blue with UV excitation and orange with green [540nm] excitation with Hoechst/pyronin stain, or yellow to greenish yellow with acridine orange stain).

Polychromatic erythrocytes (PCE) were scored by direct manual counting. Normochromatic erythrocytes (NCE) were scored using a semiautomated method, in which cell counts were determined by counting a subfield of approximately 1/16th of the full microscope field. Routine micronucleus frequency scores were based on approximately 10,000 NCE or 1000 PCE per sample, and the percentage of PCE among the total erythrocyte population was based on the number of PCE among approximately 10,000 or 5,000 erythrocytes.

Statistics:

The micronucleus results were tabulated as the mean frequency of micronucleated erythrocytes per 1000 cells per animal, plus or minus the standard error of the mean among animals within a treatment group.

The frequency of micronucleated cells among normochromatic erythrocytes or polychromatic erythrocytes was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group.

The percentage of polychromatic erythrocytes (%PCE) data were analyzed by a standard ANOVA to determine if significant PCE suppression or stimulation occurred.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Results indicate that the test has low sensitivity for prediction of carcinogenicity, a convincingly positive result in this assay appears to be highly predictive of rodent carcinogenicity.

The authors highlight that the chemicals that produced equivocal or negative results in the mouse peripheral blood MN test, predictivity for noncarcinogenic activity in rodents was poor.

Table 1: Micronucleated Erythrocyte Frequencies in Peripheral Blood of B6C3F1 Mice from NTP Subchronic Toxicity Study, males

Dose (g/kg)	N	MN-NCE/1000 NCE	P value	% PCE
Control	10	1.85 ± 0.18	0	3.17 ± 0.21
0.075	9	1.94 ± 0.18	0.2360	3.44 ± 0.25
0.15	10	1.57 ± 0.10	0.9342	3.42 ± 0.31
0.3	10	1.77 ± 0.10	0.6537	3.17 ± 0.21
0.6	10	1.50 ± 0.13	0.9725	3.29 ± 0.18
1.2	5	2.05 ± 0.07	0.1601	3.49 ± 0.45
Trend test		p=0.424
ANOVA				p=0.873

 

Table 2: Micronucleated Erythrocyte Frequencies in Peripheral Blood of B6C3F1 Mice from NTP Subchronic Toxicity Study, females

Dose (g/kg)	N	MN-NCE/1000 NCE	P value	% PCE
Control	10	1.35 ± 0.13	0	3.09 ± 0.23
0.075	9	1.20 ± 0.14	0.7667	3.26 ± 0.30
0.15	7	1.38 ± 0.21	0.4372	3.14 ± 0.40
0.3	9	1.23 ± 0.19	0.7053	3.54 ± 0.21
0.6	10	1.27 ± 0.05	0.6102	3.27 ± 0.26
1.2	4	1.48 ± 0.19	0.2586	4.18 ± 0.16
Trend test		p=0.236
ANOVA				p=0.195

 

 

 

Conclusions:

In this assay the test substance showed a negative result after 90 days exposure via oral gavage.

Executive summary:

In a study conducted by Witt et al (2000) the mouse peripheral blood micronucleus (MN) test was performed on samples collected from a sub-chronic toxicity study conducted by the National Toxicology Program (NTP). The incidence on polychromatic erythrocytes (PCE) provided an index of damage induced within 72 hours of sampling whereas the incidence of MN in the normocromatic erythrocytes (NCE) population at steady state provided an index of the average damage during the 30 -day period preceding sampling.

 

In a B6C3F1 mouse peripheral blood micronucleus assay, mice were treated by oral gavage with Butyraldehyde at doses of 0, 0.075, 0.15, 0.3, 0.6 and 1.2  g/kg bw. The vehicle was corn oil. Blood samples were taken at the day of termination.  

 

There was not a significant increase in the frequency of micronucleated NCE and PCE in the mouse peripheral blood micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity

 

In Vitro Studies

In a reverse gene mutation assay in bacteria conducted according to OECD TG 471 (Woods I. 2022) , strains TA1535, TA1537, TA98 and TA100 of S. typhimurium and E.coli WP2 uvrA pKM101 were exposed to Butyraldehyde in water at concentrations of 5, 16, 50, 160, 500, 1600 and 5000 µg/plate in the presence and absence of mammalian metabolic activation.  There was no evidence that n-Butyraldehyde was mutagenic under the conditions employed in the study.

This is further supported by several additional studies. In a study sponsored by the NTP, butyraldehyde was negative in the Salmonella/mammalian microsome mutagenicity assay when evaluated as a coded chemical in two test laboratories (Mortelmans et al. 1986; Dillon et al. 1988): It was tested in five tester strains (TA97, TA98, TA100, TA1535, TA1537) using the pre-incubation assay with a minimum of five non-toxic concentrations ranging from 33 to 3333 µg/plate, in the presence and absence of Arochlor-induced rat and hamster liver S-9 metabolic activation. Sasaki and Endo (1978) also reported negative mutagenicity results in S.typhimurium tester strains TA98 and TA100 in a pre-incubation assay with and without rat S-9 metabolic activation. The dose range tested was not reported. Pool BL and Wiessler M (1981) report a negative result in S. typhimurium tester strain TA1535 in a reverse gene mutation assay. Although, only one strain was tested and the study is not considered reliable.

 

There was an increase in the incidence of mutations at the HGPRT and Na/K ATPase loci in Chinese hamster V79 cells exposed to butyraldehyde at concentrations up to 648 µg/ml in the absence of metabolic activation (Brambilla et al. 1989). In studies utilizing primary rat and human hepatocytes, exposure to butyraldehyde at concentrations up to 2163 µg/ml did not result in an increase in DNA repair (Martelli et al. 1994). BASF (1999) reported a negative result when testing the structurally related isobutyraldehyde in the CHO cell HPRT forward mutation assay with and without metabolic activation under GLP conditions (OECD guideline 476), detailed information can be found in the isobutyraldehyde CAS 78-84-2 dossier.

Butyraldehyde was positive for induction of sister chromatid exchange in CHO cells when tested in the presence and absence of metabolic activation at nontoxic doses ranging from 9 to 90 µg/ml (Galloway et al 1987).

 

When tested in Chinese hamster ovary (CHO) cells, butryaldehyde did not increase the incidence of chromosomal aberrations at concentrations ranging from 59 to135 µg/ml with and without rat S-9 activation (Galloway et al 1987). In addition, butyraldehyde was negative for sister chromatid exchange in human lymphocytes when tested in the absence of metabolic activation (Obe and Beek 1979).

 

In Vivo Studies: Evaluation negative

A micronucleus assay in mice reportedly gave no evidence of clastogenic/genotoxic activity of butyraldehyde (NTP Testing Status 2000). Butyraldehyde was negative when tested in the sex-linked recessive lethal test in Drosophilia (SLRL). Male flies were injected with 10,000 ppm butyraldehyde or fed butyraldehyde in a 0.7% sucrose solution (Valencia et al. 1985).

 

Moutschen-Dahmen et al (1975, 1976) reported that male Q strain mice displayed mortality (LD₅₀ was 1140 mg/kg, i.p.), and evidence of cell degeneration, polyploidy during spermatogenesis, and chromosomal aberrations and modifications in sperm morphology when injected intraperitoneally (i.p.) with a single dose of 1 mg per animal (approximate dose 30 mg/kg) or exposed to the chemical in drinking water (2 g/L) for 50 days (total dose 15 g/kg or about 300 mg/kg bw/d)]. Meiotic anomalies consisting of degenerative nuclei, multispindle cells, and polyploidy were observed at all stages of spermatogenesis in exposed mice. This test series is considered unreliable due to severe deficiencies in methodology and in documentation.

 

Overview of Genetic toxicity studies

Test	Test System	Result	Reference	Evaluation
In-Vitro Assays
Ames	S. typh. (strains TA100, TA1535, TA1537, TA-98) and E.coli WP2 uvrA pKM101	Negative with and without metabolic activation	Woods I., 2022	Rel 1 (Test Guideline study)
Ames	S. typh. (strains TA100, TA1535, TA1537, TA-98) (TA 100, TA 102, TA 104)	Negative with and without metabolic activation	Mortelmans et al. 1986; Dillon et al. 1988	Rel 2
Ames	Salmonella typhimurium TA1535	Negative with and without metabolic activation	Pool and Wiessler 1981	Rel 3
Ames	S. typh. (strains TA98; TA100, TA1535, TA1537)	Negative with and without metabolic activation	Florin et al., 1980	Rel 3 (too low dose)
Ames	S. typh. (strains TA98; TA100)	Negative with and without metabolic activation	Sasaki and Endo 1978	Rel 4 (not available)
Gene mutations (mammalian cell)	HGPRT locus Na/K locus, Chinese hamster lung fibroblasts (V79) in vitro	Positive without metabolic activation	Brambilla et al. 1989	Rel 4 (secondary source)
Chromosomal aberrations	Chinese hamster ovary cells; in vitro	negative with and without metabolic activation	Galloway et al. 1987 (see also NTP Testing Status)	Rel 4 (secondary source)
Sister chromatid exchanges	Chinese hamster ovary cells; in vitro	Positive with and without metabolic activation	Galloway et al. 1987 (see also NTP Testing Status)	Rel 4 (secondary source)
Sister chromatid exchanges	Chinese hamster ovary cells; in vitro	Negative without metabolic activation	Obe and Beek 1979	Rel 3 (secondary source, deficiencies)
DNA repair	Primary hepatocyte culture (rat + human)	Negative	Martelli et al. 1994	Rel 4 (secondary source)
In-Vivo Assays
Chromosomal aberrations	Mouse, male, spermatogenesis, in vivo, i.p., single dose	Non reliable	Moutschen- Dahmen et al. 1975, 1976	Rel 3 (secondary literature)
Chromosomal aberrations	Mouse, male, spermatogenesis, in vivo, drinking water, 50d	Non reliable	Moutschen-Dahmen et al. 1976	Rel 3 (secondary literature)
Erythrocyte micronucleus assay	Mouse, peripheral blood micronucleus test, in vivo	Negative	NTP Testing Status. 2000	Rel 2 (secondary source)
Gene mutations SLRL	Drosophila melanogaster; in vivo (oral)	Negative	Valencia et al. 1985 (see also NTP Testing Status)	Rel 3 (secondary source)
Gene mutations SLRL	Drosophila melanogaster; in vivo (injection)	Negative	Valencia et al. 1985 (see also NTP Testing Status)	Rel 3 (secondary source)

Short description of key information:
The genotoxicity of the substance has been evaluated in an appropriate battery of studies in vitro and in vivo. Negative results are reported for bacterial reverse mutation in five Ames tests. Negative results are also reported for investigations of chromosomal aberration and SCE in mammalian cells in vitro, however positive results are reported in an additional study of SCE and also in an HPRT assay. USD assays in primary rat and human hepatocytes report negative results. In vivo, published studies in Drosophila report negative results and the result from two mouse studies are not reliable due to study design. One NTP study reports a negative result for the induction of micronuclei in mouse peripheral blood micronucleus test. The weight of evidence therefore suggests that the substance in not genotoxic in in vivo mammalian systems.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The genotoxicity of the substance has been evaluated in an appropriate battery of studies in vitro and in vivo. Negative results are reported for bacterial reverse mutation in five Ames tests. Negative results are also reported for investigations of chromosomal aberration and SCE in mammalian cells in vitro, however positive results are reported in an additional study of SCE and also in an HPRT assay. USD assays in primary rat and human hepatocytes report negative results. In vivo, published studies in Drosophila report negative results. Also, one NTP study reports a negative result for a mouse peripheral blood micronucleus test. The weight of evidence therefore suggests that the substance is not genotoxic in in vivo mammalian systems and does not require classification for genetic toxicity according to Directive 67/548/EEC or Regulation 1272/2008/EC (CLP).