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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 March 1997 - 24 April 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted according to GLP with slight deviation from current requirements: as positive control substance in the presence of S9 mix, only 2-aminoanthracene (2-AA) was tested.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon for S. typhimurium strains
trp operon for the E. coli strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: TA 98: rfa-, uvrB-, R-factor; TA 100: rfa-, uvrB-, R-factor; TA 1535: rfa-, uvrB-; TA 1537: rfa-, uvrB; WP2: trp-; uvr A-
Metabolic activation:
with and without
Metabolic activation system:
S9 liver mix prepared from Sprague-Dawley rats treated with Aroclor 1254
Test concentrations with justification for top dose:
First experiment (standard plate test, with and without metabolic activation): 0, 20, 100, 500, 2500 and 5000 µg/plate
Second experiment (preincubation test with and without metabolic activation): 0, 20, 100, 500, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility of the test item in the selected vehicle
Untreated negative controls:
yes
Remarks:
(sterility control)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with S9-mix
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
2.5 µg/plate, in DMSO, for TA 1535, TA 1537, TA 100, TA 98; 60 µg/plate, in DMSO, for E. coli WP2 uvrA
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
5 µg/plate, in DMSO, for TA 1535 and TA 100
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
9-aminoacridine
Remarks:
100 µg/plate, in DMSO, for TA 1537 Migrated to IUCLID6: (AAC)
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
10 µg/plate, in DMSO, for E. coli WP2 uvrA
Positive controls:
yes
Remarks:
(without S9-mix)
Positive control substance:
other: 4-nitro-o-phenylendiamine (NOPD)
Remarks:
10 µg/plate, in DMSO, for TA 98
Details on test system and experimental conditions:
In the standard plate test, tubes were filled with 2mL portions of soft agar and kept in a water bath at 45°C. This soft agar consisted of 100 mL agar and 10 mL amino acid solution. As amino acid solution for the soft agar was used 0.5 mM histidine and 0.5 mM biotin for TA strains and 0.5 mM tryptophan for the E. coli strain.
Then following components are added:
0.1 ml test solution or vehicle
0.1 ml fresh bacterial culture
0.5 ml S9 -mix or phosphate buffer
After mixing samples were poured onto Vogel-Bonner (minimal glucose agar plates) plate and incubated for 48 - 72 hrs in the dark at 37°C.

For the preincubation test 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL of the S9 mix were incubated at 37°C for 20 minutes. After addition of 2 mL soft agar samples were poured onto agar plates and incubated again at 37°C for 48 to 72 hrs.
For the E. coli strain, plate test differed again in mixture of amino acid solution of the soft agar, the histidine component used for the TA strains being replaced by tryptophan.
Triplicate testing is done.
After incubation, the his+ revertant colonies of bacteria were counted.
Evaluation criteria:
An assay is accepted when the following criteria are met:
1.) number of colonies in the negative control is in the historical control range
2.) no indication of bacterial contamination (checked by sterility control)
3.) number of colonies in the positive controls are in the range of historical control data
4.) titer of viable bacteria is ≥ 10 E+9/mL

Toxicity is detected by:
1.) decrease in the number of revertants
2.) titer reduction
3.) clearing or diminution of the background lawn

Precipitation:
As long as no interference between precipitation and colony counting occurs is 5 mg/plate set as maximum dose even for relatively insoluble compounds.

A test chemical is to be considered as mutagenic when:
1.) increase of number of revertant colonies is reproducible and dose-related.
2.) in at least 1 tester strain doubling of colony counts with or without S-9 mix or after adding a metabolizing system is seen.

A test chemical is to be considered as non-mutagenic when:
1.) the number of revertants is inside the range of historical negative control data in 2 experiments performed independently from each other.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
no increase in number of revertants was observed
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight decrease in revertants number reported for S. typhimurium TA 1535 at about 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation: no precipitation detected

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: tested in the standard plate and the preincubation test

Experiment 1: Standard plate-incorporation test

SPT without S9-Mix
 [mean no. of mutations/ plate]
Dosage [µg/plate] TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
Solvent control 21 118 14 26 35
20 18 120 11 31 35
100 19 127 9 27 32
500 16 113 10 27 35
2500 19 100 9 30 30
5000 18 114 10 25 30
Positive control 1109 1111 397 1227 652
SPT with S9-Mix
 [mean no. of mutations/ plate]
Dosage [µg/ plate] TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
Solvent control 22 119 13 42 43
20 17 109 14 53 36
100 18 125 12 45 39
500 19 107 11 44 40
2500 15 79 14 48 38
5000 14 90 11 42 33
Positive control 141 1066 118 1035 233

Experiment 2: Preincubation test PIT without S9-Mix
 [mean no. of mutations/ plate]

Dosage [µg/ plate] TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
Solvent control 20 158 13 33 33
20 18 162 11 32 32
100 18 154 11 33 32
500 16 129 10 32 31
2500 14 130 9 30 29
5000 12 132 10 30 28
Positive control 1278 1580 672 1079 791
PIT with S9-Mix
 [mean no. of mutations/ plate]
Dosage [µg/ plate] TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
Solvent control 20 158 11 47 42
20 19 166 12 46 39
100 17 154 13 44 33
500 15 140 11 43 34
2500 13 127 10 44 29
5000 11 134 8 40 28
Positive control 126 1081 139 1002 217
Conclusions:
Interpretation of results (migrated information): negative
CLP: not classified
DSD: not classified
Executive summary:

Butyldiethanolamine was tested in bacterial gene mutation test using Salmonella typhimurium starins TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA . The test concentrations were 20, 100, 500, 2500 and 5000 µg/plate for the standard plate test with and without S9 mix, as well as for the preincubation test with and without S9 mix. In none of the tests any mutagenic effect could be detected. Therefore the substance is to be considered as non-mutagenic in bacteria.

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:
Experimental starting day: 23 July 2014; Experimental completion day: 14 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
of Commission Regulation (EC) No. 440/2008 of 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
other: 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test.
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Economy, Trade and Industry (METI), Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable (structural and numerical aberrations in the chromosomes of exposed cells are detected).
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimum Essential Medium (MEM).
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/β-Naphthoflavone induced rat liver homogenate metabolizing system (S9)
Test concentrations with justification for top dose:
Experiment 1: 0, 50.31, 100.63, 201.25, 402.5, 805, 1610 (with S9 (2%) and without S9);
Experiment 2: 0, 100.63, 201.25, 402.5, 805, 1207.5, 1610 (with S9 (1%)) and 0, 100.63, 201.25, 402.5, 603.75, 805, 1207.5, 1610 (without S9).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Minimum Essential Medium (MEM).
- Justification for choice of solvent/vehicle: The test item was soluble in culture medium at 16.1 mg/mL in solubility checks performed in-house. The test item was accurately weighed, dissolved in culture medium and serial dilutions prepared.
The solubility of the test item was investigated in the Harlan Laboratories Ltd., Mouse Lymphoma Assay, Study number 41402073.
Untreated negative controls:
yes
Remarks:
vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
Minimum Essential Medium (MEM).
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
MMC (in the absence of S9; at 0.4 and 0.2 μg/mL for cultures in Experiment 1 and 2, respectively; dissolved in MEM). CP (in the presence of S9; 5 μg/mL in both experiments and was dissolved in DMSO).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours (with S9 mix (2%) and without of S9 mix in the Experiment 1 and with S9 mix (1%) in the Experiment 2); 24 hours (without S9 mix in the Experiment 2).
- Expression time (cells in growth medium): 20 hours (with S9 mix (2%) and without of S9 mix in the Experiment 1 and with S9 mix (1%) in the Experiment 2)
- Fixation time (start of exposure up to fixation or harvest of cells): directly after 20-h incubation in growth medium (expression) or after 24-h continuously exposure: "After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation".

SPINDLE INHIBITOR (cytogenetic assays): Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time.
STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED: The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were at least 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985) (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors (historical control data available)
Evaluation criteria:
The following criteria were used to determine a valid assay:
Negative controls:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range;
Positive controls:
All the positive control chemicals must induce a clear positive response (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix;
Criteria for determining the Study Conclusion:
test item can be classified as non-genotoxic if:
1. The number of induced chromosome aberrations in all evaluated dose groups is within the range of laboratory historical control data.
2. No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.
A test item can be classified as genotoxic if:
1. The number of induced structural chromosome aberrations is not in the range of laboratory historical control data.
And
2. Either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.
Biological relevance of the results will be considered first. Statistical methods will be used to analyze the increases in aberration data as recommended in the OECD 473 guideline. However, statistical significance will not be the only determining factor for a positive response. A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Experiment 1
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Experiment 1
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes:
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
Experiment 2
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes:
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
dose-related inhibition of mitotic index was observed (43% and 69% mitotic inhibition was achieved at 603.75 μg/mL and 805 μg/mL, respectively, in the absence of S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.
- Precipitation: No haemolysis or precipitate of the test item was observed in any exposure group at the end of the exposure period.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 6.29 to 1610 μg/mL. The maximum dose was the 10 mM maximum recommended concentration. No haemolysis or precipitate of the test item was observed in any exposure group at the end of the exposure period.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1610 μg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 1610 μg/mL. The mitotic index data are presented in Table 1. The test item induced evidence of toxicity in the 24-hour exposure group only.
The maximum dose level selected was the 10 mM concentration for the 4(20)-hour exposure groups, and for the continuous exposure group used in Experiment 2, where modest toxicity was demonstrated.

COMPARISON WITH HISTORICAL CONTROL DATA: the vehicle control value in the presence of S9 was marginally above the current historical range but it was considered acceptable (Experiment 1). Historical data are attached.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 6.29 to 1610 μg/mL. The maximum dose was the 10 mM maximum recommended concentration. No haemolysis or precipitate of the test item was observed in any exposure group at the end of the exposure period. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1610 μg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 1610 μg/mL. The mitotic index data are presented in Table 1 (attached to this file). The test item induced evidence of toxicity in the 24-hour exposure group only. The maximum dose level selected was the 10 mM concentration for the 4(20)-hour exposure groups, and for the continuous exposure group used in Experiment 2, where modest toxicity was demonstrated.

Chromosome Aberration Test - Experiment 1

The dose levels of the controls and the test item are given in the table below:

Group

Final concentration of test item n-Butyldiethanolamine CAS No.

102-79-4 (μg/mL)

4(20)-hour without S9

0*, 50.31, 100.63, 201.25, 402.5*, 805*, 1610*, MMC 0.4 *

4(20)-hour with S9 (2%)

0*, 50.31, 100.63, 201.25, 402.5*, 805*, 1610*, CP 5*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 1610 μg/mL in both exposure groups. The mitotic index data are given in Table 2. They confirm the qualitative observations in that there was no marked toxicity in either exposure group. The maximum dose level selected for metaphase analysis was the 10 mM concentration dose level (1610 μg/mL). The chromosome aberration data are given in Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the range expected for human lymphocytes, and although the vehicle control value in the presence of S9 was marginally above the current historical range, it was considered to be acceptable. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation. The polyploid cell frequency data are given in Table 8. The test item did not induce any statistically significant increases in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2

The dose levels of the controls and the test item are given in the table below:

Group

Final concentration of test item n-Butyldiethanolamine CAS No.

102-79-4 (μg/mL)

24-hour without S9

0*, 100.63, 201.25, 402.5*, 603.75*, 805*, 1207.5, 1610, MMC 0.2*

4(20)-hour with S9 (1%)

0*, 100.63, 201.25, 402.5, 805*, 1207.5*, 1610*, CP 5*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of 1610 μg/mL in the presence of S9. In the absence of S9, the maximum test item dose level with metaphases suitable for scoring was 805 μg/mL. The mitotic index data are given in Table 3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 43% and 69% mitotic inhibition was achieved at 603.75 μg/mL and 805 μg/mL respectively, in the absence of S9. In the presence of S9 no marked toxicity was observed. The maximum dose level selected for metaphase analysis was based on toxicity in the absence of S9 and the 10 mM concentration dose level (1610 μg/mL) in the presence of S9. The chromosome aberration data are given in Table 6 and Table 7. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation. The polyploid cell frequency data are given in Table 8. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Conclusions:
Interpretation of results (migrated information): negative
The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was therefore considered to be nonclastogenic to human lymphocytes in vitro.
Executive summary:

Butyldiethanolamine (CAS 102 -79 -4) was tested in a OECD 473 study for ability to induce chromosome aberrations in human lymphocytes (Harlan Laboratories, 2014; Report No. 41402072). Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group

Final concentration of test item n-Butyldiethanolamine CAS No.

102-79-4 (μg/mL)

4(20)-hour without S9

0, 50.31, 100.63, 201.25, 402.5, 805, 1610

4(20)-hour with S9 (2%)

0, 50.31, 100.63, 201.25, 402.5, 805, 1610

24-hour without S9

0, 100.63, 201.25, 402.5, 603.75, 805, 1207.5, 1610

4(20)-hour with S9 (1%)

0, 100.63, 201.25, 402.5, 805, 1207.5, 1610

All vehicle (MEM) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments. The test item was non-toxic in the 4(20)-hour exposure groups of Experiment and Experiment 2 and the maximum dose level was the maximum recommended dose level (1610 μg/mL) in these exposure groups. The 24-hour exposure group did demonstrate dose related toxicity and the dose range included a dose level that induced greater than optimum toxicity.

In conclusion, the test item, n-Butyldiethanolamine CAS No. 102-79-4 was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July 2014 to 27 August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
other: "Kanpoan No. 287 - Environment Protection Agency"; "Eisei No. 127 - Ministry of Health and Welfare"; "Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry".
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin В (2.5 µg/mL) and 10 % donor horse serum (giving R10 media). RPMI 1640 with 20 % donor horse serum (R20) and without serum (R0) are used during the course of the study.
- Properly maintained: yes.
- Periodically checked for Mycoplasma contamination: yes. Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically checked for karyotype stability: not reported.
- Periodically "cleansed" against high spontaneous background: yes. The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.
Additional strain / cell type characteristics:
other: - 3.7.2c
Metabolic activation:
with and without
Metabolic activation system:
The S9 microsomal fraction prepared from Sprague Dawley male rats induced with Phenobarbital/β-Naphthoflavone at 80/100 mg/kg/day (2% of final concentration in the Preliminary Experiment and Experiment 1; 1% of final concentration in the Experiment 2)
Test concentrations with justification for top dose:
- Preliminary Cytotoxicity Test: 0, 6.29, 12.58, 25.16, 50.31, 100.63, 201.25, 402.5, 805 and 1610 µg/mL;
- Main Test (Experiment 1 and Experiment 2 (4-hour)): 100.63, 201.25, 402.5, 805, 1207.25, and 1610 µg/mL;
- Main Test (Experiment 2 (24-hour)): 50, 100, 200, 300, 400, and 500 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI 1640 medium without serum (R0)
- Justification for choice of solvent/vehicle: the test item was soluble in culture media at 1610 µg/mL in solubility checks performed in-house.
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 1640 medium without serum (R0)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
EMS: at 400 μg/mL and 150 μg/mL for Experiment 1 and Experiment 2 (without S9 mix); CP at 2 μg/mL (with S9 mix). The positive controls were formulated in DMSO.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours (with and without of S9 mix in the Preliminary Experiment and in the Experiment 1 and with S9 mix in the Experiment 2); 24 hours (without S9 mix in the Experiment 2).
- Expression time (cells in growth medium): 48 hours (all experiments)
- Selection time (if incubation with a selection agent): 24 hours (on Day 2 of the experiments)
- Fixation time (start of exposure up to fixation or harvest of cells): Microtitre plates were scored using a magnifying mirror box after ten to fourteen days' incubation at 37 °С with 5% CO2 in air.

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF CELLS EVALUATED: for mutant frequency 2000 cells /well in selective medium and for viability 2 cells/ well in non-selective medium (96-well microtitre plates were used).

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: Relative Suspension Growth (% RSG)
Evaluation criteria:
Acceptability of the Assay
A mutation assay is considered acceptable if it meets the following criteria (the current recommendations of the IWGT will be considered):
1. The majority of the plates, for either viability (%V) or TFT resistance are analyzable for each experiment.
2. The absolute viability (%V) at the time of mutant selection of the solvent controls is 65 to 120 %.
3. The total suspension growth of the solvent control following 4 hour treatment, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24 hour treatment the total suspension growth should be in the range of 32 to 180.
4. The vehicle control mutant frequency should be in the range of 50 - 170 x 10E6 cells. Experiments where the vehicle control values are markedly greater than 200 x 10E6 mutant frequency per survivor are not acceptable and will be repeated.
5. Positive control chemicals (EMS and CP) should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control. The positive controls should ideally yield an absolute increase in total MF, that is an increase above spontaneous background MF (an induced MF [IMF]), of at least 300 x 10E6 cells.
6. The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures i.e. the relative total growth (RTG) and percentage relative suspension growth (%RSG) should be greater than 10 % of the concurrent selective control group.
7. The highest concentration of the test item should be 10 mM or 5000 µg/mL, unless limited by toxicity or solubility of the test item. If toxicity occurrs, the highest concentration should lower the relative total growth (RTG) and/or %RSG to approximately 10 to 20 % of survival. If precipitation is noted, the highest analyzed concentration should be the lowest concentration where precipitation is observed by the naked eye.
Statistics:
The experimental data was analyzed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W.D. et al., 1989):
ROBINSON, W.D. et al (1989) Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp 102-140.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1207.25 and 1610 µg/mL (as indicated by the % RSG and RTG values)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at > 600 µg/mL (as indicated by the %RSG and RTG values); modest reductions in viability (%V)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media
- Effects of osmolality: When the test item was dosed into media the osmolality did not increase by more than 50 mOsm.
- Precipitation: Precipitate of test item was not observed at any of the dose levels.

RANGE-FINDING/SCREENING STUDIES:
There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups, with the greatest reductions observed in the 24-hour exposure group. Precipitate of the test item was not observed at any of the dose levels. In the subsequent Mutagenicity Test, the maximum dose level was the 10 mM limit dose of 1610 μg/mL for the 4-hour exposure groups, and limited by test item-induced toxicity for the 24-hour exposure groups.

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle controls (R0 medium) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: There was no evidence of any reductions in viability (%V), therefore indicating that residual toxicity had not occurred in either of the exposure groups;
- Experiment 2: There was also evidence of modest reductions in viability (%V) in the 24-hour exposure group in the absence of metabolic activation, therefore indicating that residual toxicity had occurred.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Experiment 1 and Experiment 2 (4-hour treatment with S9 mix)

Preliminary Cytotoxicity Test

The results for the Relative Suspension Growth (%RSG) were as follows:

Dose

% RSG (-S9)

% RSG (+S9)

% RSG (-S9)

4-Hour Exposure

4-Hour Exposure

24-Hour Exposure

0

100

100

100

6.29

89

95

105

12.58

80

104

107

25.16

80

118

109

50.31

79

99

101

100.63

77

97

108

201.25

73

93

100

402.5

62

84

42

805

51

71

0

1610

21

30

0

Main Test (summary of results)

Experiment 1

4-Hour (-S-9)

4-Hour (+S-9)

Treatment

(µg/mL)

%RSG

RTG

MF§

Treatment (µg/mL)

%RSG

RTG

MF§

0

100

1.00

144.66

0

100

1.00

140.46

25.16

97

25.16

103

50.31

105

50.31

98

100.63

96

1.08

126.42

100.63

108

1.17

115.89

201.25

98

0.98

142.74

201.25

99

1.21

102.85

402.5

88

0.95

148.43

402.5

88

1.02

137.70

805

77

0.90

125.85

805

79

0.93

117.93

1207.25

52

0.54

138.22

1207.25

59

0.60

152.20

1610

33

0.39

110.88

1610

33

0.32

169.69

Linear trend

NS

Linear trend

*

EMS

400

72

0.52

896.50

CP

2

63

0.37

1320.32

Experiment2

24-Hour (-S-9)

4-Hour (+S-9)

Treatment (µg/mL)

%RSG

RTG

MF§

Treatment (µg/mL)

%RSG

RTG

MF§

0

100

1.00

162.69

0

100

1.00

126.59

25

100

25.16

89

 

50

101

1.16

115.49

50.31

94

 

100

98

0.88

185.01

100.63

88

0.90

90.43

200

92

1.07

126.42

201.25

87

0.97

110.02

300

73

0.95

132.56

402.5

96

0.90

103.30

400

35

0.48

155.58

805

106

1.03

102.96

500

7

0.14

219.91

1207.25

54

0.54

95.01

600

1

 

 

1610

31

0.29

106.32

700

0

 

 

 

 

 

 

800

0

 

 

 

 

 

 

Linear trend

NS

Linear trend

NS

EMS

CP

150

50

0.31

1229.56

2

57

0.439

742.10

MF§ = 5-TFT resistant mutants/ 10E6 viable cells 2 days after treatment

NS = Not significant

EMS = Ethylmethanesulphonate

CP Cyclophosphamide

Ø = Not plated for viability or 5-TFT resistance

Experiment 1

There was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any reductions in viability (%V), therefore indicating that residual toxicity had not occurred in either of the exposure groups. Acceptable levels of toxicity were seen with both positive control substances.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell, at any of the dose levels (including the 10 mM limit dose level) in the absence of metabolic activation. A very modest but statistically dose related (linear-trend) increase in mutant frequency was observed in the presence of metabolic activation. However, the GEF was not exceeded at any of the dose levels (including the 10mM limit dose), and the mutant frequency values observed would have been considered acceptable for vehicle controls. The response was therefore considered to be artefactual and of no toxicological significance. Precipitate of the test item was not observed at any of the dose levels.

Experiment 2

As was seen previously, there was evidence of marked toxicity in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values. There was also evidence of modest reductions in viability (%V) in the 24-hour exposure group in the absence of metabolic activation, therefore indicating that residual toxicity had occurred. Based on RTG values observed, optimum levels of toxicity were considered to have been achieved in the absence of metabolic activation. The excessive toxicity observed at and above 600 μg/mL in the absence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a marked effect on the toxicity of the test item. The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell, at any of the dose levels, including the 10 mM limit dose level in the presence of metabolic activation, and a dose level that induced optimum levels of toxicity in the absence of metabolic activation. Precipitate of test item was not observed at any of the dose levels.

Conclusions:
Interpretation of results (migrated information): negative
The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells
Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of Butyldiethanolamine on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line (OECD 476). Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated at up to ten dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for viability and expression of mutant colonies were as follows: 100.63, 201.25, 402.5, 805, 1207.25, 1610 µg/mL (Experiment 1 with and without S9; Experiment 2 with S9) and 50, 100, 200, 300, 400, 500 µg/mL (Experiment 2 without S9).

The maximum dose level used in the Mutagenicity Test was the 10 mM limit dose of 1610 μg/mL for the 4-hour exposure groups, and limited by test item-induced toxicity for the 24-hour exposure groups. Precipitate of the test item was not observed at any of the dose levels during the course of the study. The vehicle controls (R0 medium) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

Butyldiethanolamine did not induce any toxicologically significant dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment. Therefore, Butyldiethanolamine is considered to be non-mutagenic under the conditions of the test.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Mutagenicity in bacteria

In a key study, Butyldiethanolamine was tested in bacterial gene mutation test using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA (according to OECD 471; BASF, 1999; Report No. 40M0580/964324). The test concentrations were 20, 100, 500, 2500 and 5000 µg/plate for the standard plate test with and without S9 mix, as well as for the preincubation test with and without S9 mix. In none of the tests any mutagenic effect could be detected. Therefore the substance is to be considered as non-mutagenic in bacteria.

In a supporting study, n-butyldiethanolamine was evaluated in the Ames/Salmonella-E. coli Reverse Mutation Assay using Salmonella typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102 and Escherichia coli tester strain (WP2 uvrA) , in the presence and absence of an exogenous metabolic activation system (S9) (according to guidelines of different regulatory bodies; Pharmakon USA, 1996). DMSO served as solvent (negative control). Based upon the results of the prescreen, n-butyldiethanolamine was evaluated in triplicate cultures in all six tester strains, at doses of 50.0, 167, 500, 1670, 5000 and 10,000 µg/plate with and without S9, under both treatment conditions.

Revertant frequencies for all doses of n-butyldiethanolamine in all tester strains with S9, under both treatment conditions, approximated or were less than those observed in the concurrent negative control cultures. Similar results were observed in the absence of S9 in strains TA1535, TA100, TA102 and WP2uvrA under plate incorporation conditions, and in strains TA1535, TA98, TA100, TA102 and WP2 uvrA under liquid pre-incubation conditions. Statistically significant increases in revertant frequencies, to approximately 1.6- to 2.1-fold control values, were observed in strain TA98 at doses of 500 and 1670 µg/plate without S9 under plate incorporation conditions, and in strain TA1537 at doses of 50.0 and 167 µg/plate without S9 using liquid pre-incubation conditions. However, these increases were not dose dependent, and all observed revertant frequencies were within acceptable historical negative control values. A 1.6-fold increase in revertant frequency (exceeding acceptable historical negative control values) also was observed in strain TA1537 at a dose of 50.0 µg/plate without S9, but this increase was neither statistically significant nor dose dependent.

Therefore, n-butyldiethanolamine was re-evaluated under identical conditions, without S9 only, in strain TA98 using plate incorporation conditions, and in strain TA1537 using both treatment conditions. Inhibited growth again was observed in both tester strains at the highest 2-3 doses evaluated under both treatment conditions, and the test article again was freely soluble at all doses evaluated. Revertant frequencies for all doses of n-butyldiethanolamine in both tester strains under both treatment conditions approximated or were less than control values. Thus, the slight increases observed in strains TA98 and TA1537 in the original assays are considered to be statistical aberrations due to random fluctuation of the spontaneous revertant frequencies. All positive and negative control values in all assays were within acceptable ranges.

Therefore, the results for n-butyldiethanolamine were negative in the Ames/Salmonella-E. coli Reverse Mutation Assay, using liquid pre-incubation and plate incorporation treatments, under the conditions, and according to the criteria, of the test protocol.

In vitro Mammalian Cell Gene Mutation Test

The study was conducted according to a method that was designed to assess the potential mutagenicity of Butyldiethanolamine on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line (OECD 476; Harlan Laboratories, 2014, Project No. 41402073). Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9). In Experiment 2, the cells were treated at up to ten dose levels using a 4-hour exposure group in the presence of metabolic activation (1 % S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for viability and expression of mutant colonies were as follows: 100.63, 201.25, 402.5, 805, 1207.25, 1610 µg/mL (Experiment 1 with and without S9; Experiment 2 with S9) and 50, 100, 200, 300, 400, 500 µg/mL (Experiment 2 without S9).

The maximum dose level used in the Mutagenicity Test was the 10 mM limit dose of 1610 μg/mL for the 4-hour exposure groups, and limited by test item-induced toxicity for the 24-hour exposure groups. Precipitate of the test item was not observed at any of the dose levels during the course of the study. The vehicle controls (R0 medium) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

Butyldiethanolamine did not induce any toxicologically significant dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment. Therefore, Butyldiethanolamine is considered to be non-mutagenic under the conditions of the test.

In vitro Chromosome Aberration Test

Butyldiethanolamine (CAS 102 -79 -4) was tested in a OECD 473 study for ability to induce chromosome aberrations in human lymphocytes (Harlan Laboratories, 2014; Report No. 41402072). Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group

Final concentration of test item n-Butyldiethanolamine CAS No.

102-79-4 (μg/mL)

4(20)-hour without S9

0, 50.31, 100.63, 201.25, 402.5, 805, 1610

4(20)-hour with S9 (2%)

0, 50.31, 100.63, 201.25, 402.5, 805, 1610

24-hour without S9

0, 100.63, 201.25, 402.5, 603.75, 805, 1207.5, 1610

4(20)-hour with S9 (1%)

0, 100.63, 201.25, 402.5, 805, 1207.5, 1610

All vehicle (MEM) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments. The test item was non-toxic in the 4(20)-hour exposure groups of Experiment 1 and Experiment 2 and the maximum dose level was the maximum recommended dose level (1610 μg/mL) in these exposure groups. The 24-hour exposure group did demonstrate dose related toxicity and the dose range included a dose level that induced greater than optimum toxicity.

In conclusion, the test item, n-Butyldiethanolamine CAS No. 102-79-4 was considered to be non-clastogenic to human lymphocytes in vitro.


Justification for selection of genetic toxicity endpoint
No study is selected since all study results are negative.

Short description of key information:
- OECD 471 (Ames test): negative, with and without S9.
- OECD 476 (Mouse Lymphoma Assay): negative, with and without S9;
- OECD 473 (Chromosome Aberration Test): negative, with and without S9.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Butyldiethanolamine did not induce genetic toxicity in all available tests. No mutagenic activity was observed in bacterial strains of Salmonella typhimurium (Ames Test; OECD 471) and in mouse lymphoma cell line L5178Y (OECD 476). The substance was non-clastogenic to human lymphocytes in vitro (OECD 473). Therefore, in accordance with Regulation (EC) 1272/2008, the substance does not need to be classified and labelled for this endpoint.