glyoxal … %; ethandial … %

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity of Glyoxal in vitro was assessed in three different test systems performed according to OECD guideline methods. A Salmonella typhimurium reverse mutation assay (Ames Standard Plate Test) was conducted according to the OECD TG 471, using the TA1535, TA1537, TA98, TA100 and TA102 strains (BASF 40M0496/014142). Data from a further study (Pharma Research Toxicology 84.0205) were added for support since in this study, E. coli WP2 uvrA also was tested in addition to the S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538. Glyoxal was tested for clastogenicity in the in vitro mammalian chromosome aberration test according to OECD TG 473, using cultured Chinese hamster ovary (CHO) cells (BASF SE 729/211-D6172). The mutagenic potential of Glyoxal 40% aqueous solution was assessed in the in vitro mammalian cell forward gene mutation assay according to OECD TG 476 using L5178Y mouse lymphoma cells (BASF SE 729/213-D6173).

Glyoxal possesses a mutagenic/genotoxic potential in these in vitro tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

(1997)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(May 19, 2000)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B 61
- Purity test date: October 31, 2001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Glyoxal as 40% aqueous solution is stable for 6 months. The stability of the test substance as such and throughout the study period had been verified by reanalysis
- Solubility and stability of the test substance in the solvent/vehicle: the stability of the test substance (aqueous solution) at room temperature in the vehicle water also had been determined analytically in the course of the determination of the reanalysis

Target gene:

All strains are mutants from histidine auxotrophy (his) to histidine prototrophy (his+)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: defect in the excision repair system (uvrB)

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: AT base pair at the primary reversion site

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction from the liver of male Sprague-Dawley rats treated with AROCLOR 1254 mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer)

Test concentrations with justification for top dose:

- Test concentrations in the first experiment (TA 1535, TA 100, TA 102, TA 1537, TA 98): 0, 52, 260, 1300, 6500 and 13000 µg/plate
- Test concentrations in the second experiment (TA 100, TA 102): 0, 1000, 2000, 3000, 4000, 5000 µg/plate
- The concentrations tested refer to the aqueous test substance and not to the active ingredient

Vehicle / solvent:

Due to the good solubility of the test substance in water, water was selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(without S9 mix)

Positive control substance:

other: N-methyl-N´-nitro-N-nitrosoguanidine (MNNG)

Remarks:

(in DMSO at 5 µg/plate, in TA 1535 and TA 100)

Positive controls:

yes

Remarks:

(without S9 mix)

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD)

Remarks:

(in DMSO at 10 µg/plate; in TA 98)

Positive controls:

yes

Remarks:

(without S9 mix)

Positive control substance:

9-aminoacridine

Remarks:

(in DMSO at 100 µg/plate; in TA 1537) Migrated to IUCLID6: (AAC)

Positive controls:

yes

Remarks:

(without S9 mix)

Positive control substance:

mitomycin C

Remarks:

(in DMSO at 0.25 µg/plate; in TA 102) Migrated to IUCLID6: (Mit. C)

Positive controls:

yes

Remarks:

(with S9 mix)

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

in DMSO at 2.5 µg/plate in TA 1535, TA 100, TA 1537, TA 98; in DMSO at 3.0 µg/plate in TA 102

Details on test system and experimental conditions:

Standard plate test (plate incorporation method, based on Ames et al., Mut. Res., 31, 347-364, 1975 and Proc. Nat. Acad. Sci. USA, 70, 782-786, 1973):
- Soft agar was prepared from 100 mL agar (0.6% agar + 0.6% NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants consisting of 0.5 mM histidine and 0.5 mM biotin);
- Two ml portions of the soft agar were transferred into test tubes, which were then kept in a water bath at 45 °C;
- In each tube, 0.1 mL test solution or vehicle, 0.1 mL fresh bacterial culture and 0.5 mL of either S9 mix or phosphate buffer were added;
- The content of each tube was mixed and poured onto Vogel-Bonner agar plates (minimal glucose agar plates);
- The plates were incubated at 37 °C for 48 to 72 hours;
- Three test plates per test concentration or control were used;
- At the end of the incubation time, the bacterial colonies (his+ revertants) were counted;
- Cytotoxicity was determined in all test groups and both experiments, with and without S-9 mix; the plates were examined for decrease in the number of revertants, diminution of background lawn, and reduction in titer;
- The titer was determined only in the experimental parts with S-9 mix both for the negative controls (vehicle only) and for the 2 highest doses in both experiments;
- Precipitation of the test material was recorded.

Evaluation criteria:

The test chemical is considered positive if the following criteria is met:
Dose-related and reproducible increase in the number of revertant colonies, i .e. about doubling of the spontaneous mutation rate in at least one tester strain either with or without S-9 mix;
The test chemical is considered negative if the following criteria is met:
The number of revertants for all tester strains is within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

The mean number of revertant colonies per plate and the standard deviations were determined for all dose groups as well as for the positive and negative (vehicle) controls in both experiments.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Experiment 1: an increase in the number of mutant colonies was observed at 1300 µg/plate (factor 2.3) and at 6500 µg/plate (factor 2.2). Experiment 2: an increase in the number of mutant colonies was observed at 2000 and 3000 µg/plate

Cytotoxicity / choice of top concentrations:

other: Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Experiment 1: an increase in the number of mutant colonies was observed at 1300 µg/plate (factor 2.2). Experiment 2: an increase in the number of mutant colonies was observed from 1000 µg/plate (factor 2.8) onwards.

Cytotoxicity / choice of top concentrations:

other: Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Experiment 1: a slight increase in the number of mutant colonies was observed at 1300 µg/plate (factor 2.0). Experiment 2: a slight increase in the number of mutant colonies was seen in the second experiment at 2000 µg/plate (factor 1.6).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Experiment 1: a slight increase in the number of mutant colonies was observed at 1300 µg/plate (factor 2.0). Experiment 2: a slight increase in the number of mutant colonies was observed at 2000 µg/plate (factor 1.7).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No increase in the number of mutant colonies was observed (factors ranging from 0.1 to 1.3 in absence of metabolic activation and from 0.3 to 1.7 in presence of metabolic activation).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No increase in the number of mutant colonies was observed (factors ranging from 0.1 to 1.3 in absence of metabolic activation and from 0.3 to 1.7 in presence of metabolic activation).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No increase in the number of mutant colonies was observed (factors ranging from 0.1 to 1.3 in absence of metabolic activation and from 0.3 to 1.7 in presence of metabolic activation).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2001-09-19 to 2001-10-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1997)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

(1998)

Qualifier:

according to guideline

Guideline:

other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests (1995)

GLP compliance:

yes

Type of assay:

other: Chromosome Aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B 61
- Purity test date: October 31, 2001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Glyoxal as 40% aqueous solution is stable for 6 months. The stability of the test substance as such and throughout the study period had been verified by reanalysis.
- Solubility and stability of the test substance in the solvent/vehicle: the stability of the test substance (aqueous solution) at room temperature in the vehicle water also had been determined analytically in the course of the determination of the reanalysis

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO cells (a permanent line originally derived from a biopsy of Chinese hamster ovary) is characterized by a low number of chromosomes, making scoring relatively easy. The modal number of chromosomes in the CHO cells used in the testing laboratory (Covance Lab. Ltd) was 21.

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction from the liver of male Sprague-Dawley rats treated with AROCLOR 1254 mixed a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer)

Test concentrations with justification for top dose:

Following concentrations referring to the active ingredient were tested, both with and without S-9 mix:
13.06, 16.33, 20.41, 25.51, 31.89, 39.86, 49.82, 62.28, 77.85, 97.31, 121.6, 152.0, 190.1, 237.6, 297.0, 371.2, 464.0, 580.0 µg/mL

Vehicle / solvent:

purified water

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Remarks:

(with S9 mix)

Positive control substance:

cyclophosphamide

Remarks:

tested at 3.125, 6.25 and 12.5 µg/mL

Positive controls:

yes

Remarks:

(without S9 mix)

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

tested at 0.0625, 0.125, 0.25 µg/mL

Details on test system and experimental conditions:

- The Glyoxal test series (with and without S-9 mix) were accompanied by negative solvent controls (solvent: water), positive controls, and further were compared to a laboratory historical (normal) control range;
- For all test groups except the negative controls, duplicate cell cultures were used; for the negative controls, 4 cultures per test group were used;
- The 3 hour-treatment of the cultures was followed by a post-exposure incubation period of 17 hours prior harvesting. About 2 hours prior to harvesting, 1 µg/ml Colchicine was added to each flask to arrest dividing cells in metaphase;
- The cultures were subjected to a series of centrifugation, resuspension and fixation steps, chromosome spreading was enhanced, and finely cell preparations were transferred onto clean microscope slides, which were dried, stained (Giemsa) and mounted with coverslips;
- Cell number was determined using a Coulter counter;
- Concentration levels to be analysed were selected from the series of test concentrations on the basis of cytotoxicity findings; the highest level to be selected should be the one with at least 50% relative cell growth;
- One hundred well-spread metaphases per replicate of each culture were evaluated for chromosomal aberrations. Only cells displaying 19 to 23 chromosomes were acceptable for examination; cells with > 23 chromosomes showing polyploidy, endoreduplication or hyperdiploidy were noted and recorded separately;
- Structural chromosome aberrations were classified according to the ISCN scheme (An International System for Human Cytogenetic Nomenclature, Editor Felix Mitelman S Karger, Switzerland, 1995). The aberrant cells in each culture were categorised as follows:
- Cells with structural aberrations including gaps
- Cells with structural aberrations excluding gaps
- Polyploid, endoreduplicated or hyperdiploid cells.

Evaluation criteria:

The criteria for a positive response were as follows:
- The proportions of cells with structural aberrations at one or more concentration exceeds the normal range in both replicate cultures,
- A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at these doses.

Statistics:

The findings and differences between treated and control results were assessed statistically.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

(Glyoxal induced chromosome aberrations in cultured Chinese hamster ovary (CHO) cells in the absence and presence of metabolic activation (S-9) when tested to concentrations up to 580.0 µg/mL, which corresponded to 10 mM.)

Cytotoxicity / choice of top concentrations:

other: Without S9 mix, cytotoxicity became evident from 25.51 µg/ml; with S9 mix, cytotoxicity became evident from 237.6 µg/ml. Thus, following concentrations were selected for cytogenic evaluation: 25.51, 190.1 and 580.0 µg/ml in absence of S9 mix, and 190.1, 3

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2001-09-10 to 2001-12-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1997)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

(1998)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B 61
- Purity test date: October 31, 2001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Glyoxal as 40% aqueous solution is stable for 6 months. The stability of the test substance as such and throughout the study period had been verified by reanalysis.
- Solubility and stability of the test substance in the solvent/vehicle: the stability of the test substance (aqueous solution) at room temperature in the vehicle water also had been determined analytically in the course of the determination of the reanalysis

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase locus (HPRT).

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction from the liver of male Sprague-Dawley rats treated with AROCLOR 1254 mixed a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer)

Test concentrations with justification for top dose:

CYOTOXICITY:
In the cytotoxicity range-finding experiment, 10 concentrations were tested in the absence and presence of S-9 mix, separated by two-fold intervals; the test concentrations ranged from 3.13 μg/ml to 1600 μg/mL.

MAIN TEST (comprising 3 experiments):
The test concentrations selected for the main test and expressed in terms of the active ingredient.
Experiment 1:
With S9 mix: 0, 25, 50, 100, 125, 150, 175, 200, 400 µg/mL
Without S9 mix: 0, 25, 50, 100, 200, 250, 300, 350, 400 µg/mL
Experiment 2:
With S9 mix: 0, 25, 50, 100, 200, 250, 300, 350, 400 µg/mL
Without S9 mix: 0, 100, 200, 250, 300, 325, 350, 375, 400 µg/mL
Experiment 3 (confirmatory test, without S-9 mix):
0, 100, 200, 250, 275, 300, 325, 350, 375, 400 µg/mL

Vehicle / solvent:

purified water

Negative solvent / vehicle controls:

yes

Remarks:

(water)

Positive controls:

yes

Remarks:

(with S9 mix)

Positive control substance:

benzo(a)pyrene

Remarks:

tested at 2.00 and 3.00 µg/mL

Positive controls:

yes

Remarks:

(wthout S9 mix)

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

tested at 0.10 and 0.15 µg/mL

Details on test system and experimental conditions:

- A cytotoxicity range-finding test was conducted with and without S9 mix for determination of the adequate test levels for the mutagenicity assay;
- The mutagenicity assay consisted of 2 experiments performed in the absence and presence of S-9 mix, and a third, confirmatory experiment conducted in absence of S-9 mix;
- The Glyoxal test series (+ and - S9) were accompanied by negative solvent and by positive controls; the data were further compared to a laboratory historical (normal) control range;
- Defined amounts of cells in medium were placed in sterile 50 ml centrifuge tubes containing the test material or thepositive control substance; where necessary, S9 mix was added;
- Each treatment, was performed in duplicate cultures; single cultures were used for testing of the positive control substances;
- After a treatment period of 3 hours at 37 °C, the cells densities were determined and the cells were either transferred to flasks for growth through the expression period or diluted to be plated for survival;
- For viability/survival plating, the plates were incubated (37°C, 5% CO2) for a period of 10 to 11 days;
- For 6TG resistance plating, the plates were incubated and the number of 6-TG resistant mutants/10E+6 viable cells was determined after 7 days following treatment; clones were identified by eye using background illumination and counted.

Evaluation criteria:

The test substance is mutagenic if:
- The validity criteria for the assay are fulfilled,
- The mutant frequency at one or more doses is significantly greater than that of the negative control,
- There is a significant dose-relationship as indicated by the linear trend analysis,
- The effects are reproducible.

Statistics:

Mutant frequencies and differences from control were assessed statistically.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Under the test conditions used, Glyoxal tested up to its limit of toxicity showed some evidence of mutagenic activity in L5178Y Mouse Lymphoma Cells in the absence of S-9. No mutagenic activity was seen in the presence of S-9.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the cytotoxicity range-finding experiment, cytotoxicity in the absence of S-9 mix was clearly observed above 200 µg/ml (relative survival 41% versus 100% for the solvent control), in fact, at the 3 highest test concentrations of 400, 800 and 1600 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1:
In the absence of S9 mix, no statistically significant increases in mutant frequency were observed at any dose level analysed in the first experiment; however, a statistically significant dose-related linear trend was evident. In the presence of S9 mix, no statistically significant increases in mutant frequency were observed at any dose level analysed in the first experiment. No dose-related linear trend could be evidenced.

Experiment 2:
In the absence of S9 mix, statistically significant increases in mutant frequency were observed at the highest two doses analysed in the present experiment (250 and 300 μg/ml). A statistically highly significant dose-related linear trend was evident. In the presence of S9 mix, no statistically significant increases in mutant frequency were observed at any dose level analysed in the present experiment. Only a weak dose-related linear trend could be evidenced. As no statistically significant increases in mutant frequency had been seen, the linear trend observed here was not considered to be of biological significance. Only in this experiment, an increased heterogeneity was reported between the replicate cultures treated with 250 µg/ml test substance, in the absence of S9 mix. However, the respective mutant frequencies both were considerably above solvent control value (29.20 and 10.84 mutants/106 viable cells, versus 4.26 for control) and did not have an excessive impact on the overall heterogeneity for mutation in this experiment. In this experiment, the overall heterogeneity values for survival (in absence of S9 mix) and for mutation (in presence of S9 mix), were reported to be slightly higher as expected. However, referring to survival, the heterogeneity seen at each dose level was within normal limits. Referring to mutation, heterogeneity only was due to the replicates of the negative solvent controls, and values furthermore were close to historical mean value. Therefore, the levels of heterogeneity for survival and mutation were not considered to affect to validity of the study.

Experiment 3 (confirmatory test):
In the absence of S9 mix, statistically significant increases in mutant frequency were observed at the highest doses of 275, 325 and 375 μg/ml in the present experiment. Moreover, in this confirmatory test, increased mutant frequencies compared to the concurrent solvent controls, were observed throughout the dose range. A statistically highly significant dose-related linear trend was evident.

Results of experiment 1:

Mutant frequencies (6-TG resistant mutants/10^6cells 7 days after treatment with Glyoxal)
- S-9			+ S-9
Concentration^RM(µg/mL)	MF		Concentration^RM(µg/ml)	MF
0	6.69		0	7.02
25	5.81		25	6.69
50	7.85		50	6.51
100	6.66		100	6.02
200	11.25		125	7.24
250	9.34		150	6.52
300	9.01		175	10.58
350	9.45		200	7.58
Linear trend *		Linear trend not significant
Positive control (NQO)			Positive control (BP)
0.1	21.28		2	68.70
0.15	37.20		3	135.68
^RM, only concentrations retained for analysis
^*, **, ***, test for linear trend significant at 5%, 1% 0.1% respectively

Results of experiment 2:

Mutant frequencies (6-TG resistant mutants/10^6cells 7 days after treatment with Glyoxal)
- S-9		+ S-9
Concentration^RM(µg/mL)	MF	Concentration^RM(µg/ml)	MF
0	4.26	0	8.06
100	5.82	25	2.84
200	9.08	50	4.23
250	19.87*	100	3.82
300	19.20*	200	11.60
		250	12.35
Linear trend ***
Positive control (NQO)		Positive control (BP)
0.1	28.64	2	47.84
0.15	43.24	3	84.40
^RM, only concentrations retained for analysis
^*, **, ***, test for linear trend significant at 5%, 1% 0.1% respectively

Results of experiment 3 (confirmatory test):

Mutant frequencies (6-TG resistant mutants/10^6cells 7 days after treatment with Glyoxal)
- S-9
Concentration^RM(µg/mL)	MF
0	3.78
100	7.25
200	8.18
250	8.63
275	9.78*
300	8.86
325	10.61*
350	9.66
375	11.63
Linear trend ***
Positive control (NQO)
0.1	48.71
0.15	62.51
^RM, only concentrations retained for analysis
^*, **, ***, test for linear trend significant at 5%, 1% 0.1% respectively

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The genotoxicity of Glyoxal in vivo was assessed in a micronucleus assay (MNT) performed with mice, according to OECD 474, in a somatic cell gene mutation assay in transgenic mice (TGR) according to OECD 488 and in a UDS test using isolated rat hepatocytes according to OECD 486. The tests were conducted in compliance to GLP (BASF SE 729/215-D6172; BASF SE 729/217-D6173; BASF SE 60M0496/01X123). Additional MNT studies were considered for support (Société Francaise Hoechst 2018 MAS; Pharmakon PH 309A-AC-002-82). Two sex-linked recessive lethal (SLRL) tests using Drosophila melanogaster for detection of point mutations and small deletions in the germ line of the insect were added for support (Barnett BM 1989 and American Cyanamid Company 1983). As further support for discussion, the publications of Furihata (1985 and 1989) and Ueno (1991) having investigated glyoxal-induced in vivo UDS in the stomach of rat also were considered.

In vivo, Glyoxal showed positive results, which are restricted to the pyloric mucosa of the stomach and to the liver and were induced at high dose levels of Glyoxal (50 mg/kg bw) and unknown purity. However, no clastogenic potential could be evidenced for pure Glyoxal in modern guideline studies according to GLP, such as the micronucleus test, and the UDS assay which revealed no genotoxic potential. In a recent somatic cell gene mutation assay in transgenic mice (TGR) the test substance did not induce gene mutation in either the liver, stomach or duodenum. All in all, there is no mutagenic potential of Glyoxal in vivo germ cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Aug 2020 - 13 Sep 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

Version / remarks:

26 June 2020

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Transgenic Rodent Somatic Cell Gene Mutation Assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number: B62, 16.03.2020
- Purity: 39.8%
- Physical state, appearance: Liquid, colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (acceptable range: 15 to 25°C), under N2
- The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- The stability of the test substance formulation in vehicle (Water for injection) was confirmed after storage for 4 days under room temperature.
- Homogeneity: Given
- Expiration date: 16 March 2021
- Actual storage temperature and storage period: 20.0 to 23.6°C; 30 April to 20 September 2020 (from receipt to the final day of use)

Species:

mouse

Strain:

other: CD2-LacZ80/HazfBR (MutaMouse) [SPF]

Details on species / strain selection:

Justification for test system selection:
CD2-LacZ80/HazfBR mice are commonly used as transgenic animals, and animals of this strain are readily available in in vivo gene mutation assays.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Trans Genic Inc. (currently known as Kobe Research Institute, BioSafety Research Center Inc.)
- Age at study initiation: 9 weeks
- Weight at study initiation: 26.4 to 29.2 g
- Assigned to test groups randomly: based on their body weights on Day 1 using LATOX-F/V5 computer system package
- Housing: individually in a plastic cage (W 18.2 × D 26.0 × H 12.8 cm)
- Diet: ad libitum, pellet diet CRF-1 (lot No. 200305, Oriental Yeast)
- Water: ad libitum, tap water from water bottles
- Acclimation period: Day -8 to 1

ENVIRONMENTAL CONDITIONS
- Temperature: 22.4 - 23.9°C
- Humidity: 49 - 93%
- Air changes: 12 times/h
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 Aug 2020 To: 25 Sep 2020

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: water for injection (OTSUKA DISTILLED WATER, Otsuka Pharmaceutical Factory, Inc)
- Concentration of test material in vehicle: 40, 12, 4 mg/mL
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no.: 0B94N

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- test substance were weighed into a graduated test tube
- adding of an appropriate volume of vehicle
- test substance was dissolved by vortexing.
- further dilution to 15 mL with vehicle to prepare 4, 12, 40 mg/mL formulations, and stirred using a vortex mixer.
- Each test substance formulation was divided into 3 or 4 portions for each administration and stored in tight and light-resistant containers under room temperature until use and maximally for 3 days.
- It was confirmed that the test substance formulations had been appropriately prepared by a concentration determination method validated by the Sponsor.

RATIONALE FOR DOSE SELECTION:
The dose were selected based on a range finding study in CD2F1/Slc mice were treated with 0, 100, 400 or 1000 mg/kg bw/day test substance in water for injection over a period of 14 days.

Duration of treatment / exposure:

Negative control and test substance-treated groups: 28 d (Days 1 - 28)
Positive control group: 2 d (Day 2 - 3)

Frequency of treatment:

once daily

Post exposure period:

Manifestation period:
Negative control and test substance-treated groups: 3 d (Day 29 - 31)
Positive control: 10 d (Days 4 - 13)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control (water for injection)

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

120 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Treated: 7 males per dose
Evaluated: 6 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

N-ethyl-N-nitrosourea (ENU), contains 40% water, 1.8% AcOH
- Justification for choice of positive control(s): Considering information in the OECD test guideline and academic documents, this substance was selected
- Route of administration: intraperitoneally once daily for 2 consecutive days at about 24-hour intervals
- Dose: 100 mg/kg bw/day, applied twice at the interval of 24 hours
- Lot No.: 11-DPM-56-3
- Maunfacturer: Toronto Research Chemicals Inc.

Tissues and cell types examined:

Organs removed, weighed and macroscopic examination : liver, stomach, duodenum, kidney, heart, bladder, lymphe node (mesenteric)

Histopathological examination and extraction of genomic DNA: liver, stomach, duodenum

Other examinations:
- Individual body weights (measured on Day 1, 3, 8, 13/15, 22, 29, 31)
- Food weight per cage (measured on Day 1, 3, 8, 15, 22, 29)
- Clinical signs (twice daily during administration period, once daily afterwards)

Details of tissue and slide preparation:

DETERMINATION OF THE MUTANT FREQUENCY:

- Fixation: The liver, stomach, duodenum, kidney and lymph node (mesenteric) were fixed in an adequate volume of 10% neutral buffered formalin solution.

- Preparation of the histopathological specimens: Specimens for histopathological examination were prepared for liver, stomach and duodenum from all animals in negative control group and all test substance-treated groups. Fixed tissue samples were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H.E.) according to the routine method.

- Determination of mutation frequency:
Genomic DNA is extracted from the selected organs of mice dosed with the test substance. The target gene (lacZ) is recovered from the genomic DNA by incorporation of the transgene into a λ bacteriophage or plasmid shuttle vector. The entire volume of packaged DNA sample was added to Escherichia coli (lacZ-, galE-) suspension and mixed. The tube was incubated at room temperature for about 30 minutes to allow phage to infect E. coli. The bacteria suspension was the plated on LB agar containing P-gal for selection. When necessary, the packaging procedure was repeated until the total number of plaques per animal reached 300,000.

- Calculation of mutant frequency:
The mutant frequency in a concerned organ was calculated by dividing the number of mutant plaques by the total number of plaques.

Mutant frequency = Number of mutant plaques/Total number of plaques


SEQUENCING OF THE MUTANT PLAQUES:

- Sampling of mutant plaques:
The mutation frequency in the liver of two animals (animal no. 1103 (low dose treated animal) and animal no. 1304 (high dose treated animals)) exceeded the range of the historical data. Therfore 30 mutant plaques of the liver samples from each selection plate were collected for subsequent sequencing of the nucleotide sequence. Sequencing was conducted by the Sponsor.

- Amplification of mutant lacZ reporter genes using polymerase chain reaction (PCR):
The purified and concentrated DNA of each mutant plaque was used for two independent polymerase chain reactions in order to distinguish PCR induced errors from the true mutations.

- DNA library preparation:
The purified PCR products of all mutants from a single animal were pooled together. Separate pools were prepared from the technical PCR replicates for each animal. From each pool an individual library was prepared for sequencing. Library quality was checked on a microchip electrophoresis system.

- Next generation sequencing:
Each library was normalized to a concentration of 2 nM, and then further diluted to a final concentration of 1.6 pM. The 2x 150 bp paired-end sequencing run was started on the Illumina® MiniSeq™ System. This system uses the Illumina® sequencing by synthesis technique.

Evaluation criteria:

INTERPRETATION OF THE RESULTS:
The test result was considered to be positive, if the following criteria are met:
- The test substance-treated groups exhibits a statistically significant increase of the mutant frequency compared with the negative control.
- The mutant frequency (mean of group value) in the test substance-treated group is greater than the acceptable range calculated from the test facility’s historical data of the negative control group.

In addition, the biological relevance of the results was taken into consideration for the final judgment

VALIDITY OF STUDY
Since the following conditions were satisfied, the test was considered successfully performed:
- The mutant frequency in the positive control group markedly increases with a statistically significant difference from the negative control group.
- The mutant frequency in the negative control group should be within the acceptable range (mean ± 3SD) calculated from the historical data at BSRC.

Statistics:

The data from the negative control group and each test substance-treated group were tested by Bartlett’s test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity was determined (not significant on Bartlett’s test), then Dunnett’s multiple comparison test was performed to assess the statistical significance of differences between the negative control group and each test substance-treated group (two-sided, familywise significance level of 0.05). If there was no homogeneity (significant on Bartlett’s test), Steel’s test (two-sided, significance level of 0.05) was performed to analyze the differences.

The data on the mutant frequency from the negative control group and the positive control group were tested by F test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity of variance was determined (not significant on F test), Student’s t test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and the positive control group. If there was no homogeneity (significant on F test), Aspin-Welch’s t test (two-sided, significance level of 0.05) was performed to analyze the differences.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the dose range-finding study, in CD2F1/Slc mice treated with 0, 100, 400 or 1000 mg/kg bw/day (as active ingredient) of test substance in water for injection over a period of 14 days. No clinical signs of toxicity were observed in any of the groups. In the histopathological examination, hyperkeratosis, squamous cell hyperplasia and inflammatory cell infiltration were observed in the forestomach in the 400 and 1000 mg/kg bw/day groups. And, inflammatory cell infiltration and erosion/ulcer with penetration were observed in the forestomach or glandular stomach in the in the 1000 mg/kg bw/day group. In addition, eosinophilic change of hepatocyte was observed in the 400 and 1000 mg/kg bw/day groups. Therefore, the doses 40.0, 120, and 400 mg/kg bw/day (as active ingredient) were selected in the present study.

RESULTS OF DEFINITIVE STUDY
- Mutant frequency liver (Table 1 and 4): No statistically significant increase was observed compared with the negative control group. All mean values of the negative control and the test substance-treated groups were within the acceptable control range calculated from the historical negative control data. However, the individual values of one animal from the low dose and middle dose groups each were higher the upper value of the historical control range (mean ± 3SD). The results of an additional assay were compareable to the first resulte. A statistically significant increase of the positive control group mutant frequency was observed in both assay.
The results of the sequencing (Table 7 and 8) of the mutant plaques showed that in the 30 mutant plaques obtained from animal of the low dose group, 4 different true mutations had taken place, of which 1 mutation was present in approx. 75% of the cases. In case of the mutant analysis of the 30 mutant plaques obtained from the liver of animal of the high dose group only two different true mutations were detected, whereby also here 1 mutation was present in approx. 93% of the cases.
In conclusion, the study strongly indicates, that the observed increased mutant frequencies in the liver of the two animals more likely resulted from clonal expansion of single mutations and do not represent a true mutagenic effect of the test substance.

- Mutant frequency stomach (Table 2 and 5): No statistically significant increase was observed compared with the negative control group. All mean values of the negative control and the test substance-treated groups were within the acceptable control range calculated from the historical negative control data. However, the individual values of one animal from the high dose were higher the upper value of the historical control range (mean ± 3SD). The results of an additional showed no individual values exceeding the historical data. A statistically significant increase of the positive control group mutant frequency was observed in both assay.

- Mutant frequency duodenum (Table 3): No statistically significant increase was observed compared with the concurrent negative control group. All mean values of the negative control and the test substance-treated groups were within the acceptable control range calculated from the historical negative control data. Furthermore, they were within the non-parametric Tukey limit range calculated from the individual data of the historical negative control data. When compared to the negative control group, a statistically significant increase of the positive control group mutant frequency was observed.

- Body weight and general conditions: There were no statistically or biologically significant differences observed.

- Food consumption: There were no statistically or biologically significant differences observed.

- Organ weight and relative organ weight: In the 120 and 400 mg/kg bw/day groups, the relative liver weight statistically significantly decreased compared with the negative control group. In addition, in the 400 mg/kg bw/day group, the stomach weight, the relative stomach weight, the duodenum weight and the relative duodenum weight statistically significantly increased compared with the negative control group.

- Gross necropsy findings: In the 400 mg/kg bw/day group, thick and brown patch in the forestomach were observed in six animals. In the negative control group, black in the spleen was observed in one animal and brown nodule in the liver was observed in one animal. This findings were considered to be a spontaneous lesion because it was found in the negative control group. There were no macroscopic findings related to test substance-treatment in the abdominal, thoracic, pelvic and cranial cavities in the other of the test substance-treated groups.

- Histopathological findings (Table 6): In the 400 mg/kg bw/day group, eosinophilic change in the liver was observed in all seven animals, and erosion/ulcer, hyperkeratosis and squamous cell hyperplasia in the forestomach were observed in six animals, and inflammatory cell infiltration in the glandular stomach was observed in two animals. In the 120 mg/kg bw/day group, inflammatory cell infiltration in the glandular stomach was observed in one animal. There were no findings related to test substance-treatment in the duodenum.

Table 1: Induction of mutation in liver of transgenic mice treated with test item [Male mice dosed once a day, for 28 days (Oral administration, 3 days after final administration)]

Substance	Dose (mg/kg bw/day, p.o.)	Animal ID No.	Number of plaque forming units	Number of packagings	Number of mutants	Mutant frequency (×10^-^6)	Group Mean ± S.D. (×10^-^6)
D.W.	0	1001	494,100	1	18	36.4
		1002	594,000	2	34	57.2
		1003	325,800	2	20	61.4	54.7	±	15.6
		1004	330,300	1	16	48.4
		1005	657,900	1	29	44.1
		1006	322,200	1	26	80.7
Test item	40.0	1101	757,800	2	38	50.1
		1102	491,400	1	30	61.1
		1103	472,500	1	46	97.4	61.1	±	19.1
		1104	302,400	1	13	43.0
		1105	504,000	1	27	53.6
		1106	308,700	1	19	61.5
	120	1201	322,200	1	17	52.8
		1202	713,700	2	42	58.8
		1203	416,700	1	23	55.2	57.3	±	20.6
		1204	697,500	2	34	48.7
		1205	452,700	1	43	95.0
		1206	333,000	1	11	33.0
	400	1301	322,200	1	23	71.4
		1302	377,100	1	21	55.7
		1303	310,500	1	12	38.6	61.3	±	15.7
		1304	359,100	1	29	80.8
		1305	409,500	1	29	70.8
		1306	375,300	1	19	50.6
ENU	100	1401	371,700	1	91	244.8
		1402	423,900	1	93	219.4
		1403	361,800	1	77	212.8	208.1	±	40.1	*(S)
		1404	305,100	1	47	154.0
		1405	350,100	1	88	251.4
		1406	306,900	1	51	166.2

D.W.: Negative control (Water for injection, 10 mL/kg)

ENU: Positive control (N -ethyl-N -nitrosourea, 10 mL/kg, i.p., dose once a day, for 2 days, expression period; 10 days)

*: Significant difference from negative control (p<0.05), (S): Student's t test

 

 

Table 2: Induction of mutation in stomach of transgenic mice treated with test item [Male mice dosed once a day, for 28 days (Oral administration, 3 days after final administration)]

Substance	Dose (mg/kg bw/day, p.o.)	Animal ID No.	Number of plaque forming units	Number of packagings	Number of mutants	Mutant frequency (×10^-^6)	Group Mean ± S.D. (×10^-^6)
D.W.	0	1001	518,400	1	28	54.0
		1002	610,200	2	32	52.4
		1003	401,400	2	15	37.4	54.5	±	11.7
		1004	715,500	2	39	54.5
		1005	749,700	2	41	54.7
		1006	485,100	2	36	74.2
Test item	40.0	1101	324,000	1	12	37.0
		1102	441,000	1	31	70.3
		1103	430,200	1	21	48.8	58.5	±	15.2
		1104	305,100	1	23	75.4
		1105	690,300	2	35	50.7
		1106	683,100	2	47	68.8
	120	1201	554,400	2	31	55.9
		1202	314,100	1	16	50.9
		1203	369,900	1	10	27.0	48.2	±	13.5
		1204	519,300	2	33	63.5
		1205	322,200	1	12	37.2
		1206	419,400	1	23	54.8
	400	1301	384,300	1	16	41.6
		1302	452,700	1	37	81.7
		1303	603,000	2	29	48.1	62.6	±	19.0
		1304	472,500	1	42	88.9
		1305	360,000	1	19	52.8
		1306	446,400	1	28	62.7
ENU	100	1401	302,400	1	139	459.7
		1402	435,600	1	219	502.8
		1403	321,300	1	178	554.0	536.3	±	62.4	*(AW)
		1404	422,100	2	253	599.4
		1405	450,000	1	276	613.3
		1406	518,400	1	28	54.0

D.W.: Negative control (Water for injection, 10 mL/kg)

ENU: Positive control (N -ethyl-N -nitrosourea, 10 mL/kg, i.p., dose once a day, for 2 days, expression period; 10 days)

*: Significant difference from negative control (p<0.05), (AW): Aspin-Welch's t test

 

Table 3: Induction of mutation in duodenum of transgenic mice treated with test item [Male mice dosed once a day, for 28 days (Oral administration, 3 days after final administration)]

Substance	Dose (mg/kg bw/day, p.o.)	Animal ID No.	Number of plaque forming units	Number of packagings	Number of mutants	Mutant frequency (×10^-^6)	Group Mean ± S.D. (×10^-^6)
D.W.	0	1001	516,600	1	31	60.0
		1002	518,400	1	31	59.8
		1003	477,900	1	25	52.3	67.6	±	12.2
		1004	306,900	1	26	84.7
		1005	459,900	1	33	71.8
		1006	390,600	1	30	76.8
Test item	40.0	1101	316,800	1	21	66.3
		1102	674,100	1	48	71.2
		1103	497,700	1	40	80.4	76.6	±	10.9
		1104	415,800	1	31	74.6
		1105	361,800	1	35	96.7
		1106	569,700	1	40	70.2
	120	1201	393,300	1	39	99.2
		1202	395,100	1	23	58.2
		1203	542,700	1	35	64.5	70.1	±	15.6
		1204	420,300	1	24	57.1
		1205	577,800	1	43	74.4
		1206	463,500	1	31	66.9
	400	1301	375,300	1	28	74.6
		1302	739,800	1	50	67.6
		1303	341,100	1	27	79.2	73.5	±	9.6
		1304	574,200	1	48	83.6
		1305	349,200	1	20	57.3
		1306	380,700	1	30	78.8
ENU	100	1401	381,600	1	326	854.3
		1402	621,000	1	502	808.4
		1403	525,600	1	603	1,147.3	961.6	±	144.5	*(AW)
		1404	498,600	1	483	968.7
		1405	415,800	1	467	1,123.1
		1406	464,400	1	403	867.8

D.W.: Negative control (Water for injection, 10 mL/kg)

ENU: Positive control (N -ethyl-N -nitrosourea, 10 mL/kg, i.p., dose once a day, for 2 days, expression period; 10 days)

*: Significant difference from negative control (p<0.05), (AW): Aspin-Welch's t test

 

Table 4: Induction of mutation in liver of transgenic mice treated with the test item (Additional assay) [Male mice dosed once a day, for 28 days (Oral administration, 3 days after final administration)]

Substance	Dose (mg/kg bw/day, p.o.)	Animal ID No.	Number of plaque forming units	Number of packagings	Number of mutants	Mutant frequency (×10^-^6)	Group Mean ± S.D. (×10^-^6)
D.W.	0	1001	315,000	1	14	44.4
		1002	423,900	1	21	49.5
		1003	376,200	1	11	29.2	46.9	±	12.0
		1004	333,000	1	15	45.0
		1005	599,400	1	28	46.7
		1006	330,300	1	22	66.6
Test item	40.0	1101	732,600	1	40	54.6
		1102	484,200	1	15	31.0
		1103	526,500	1	47	89.3	51.8	±	23.2
		1104	325,800	1	14	43.0
		1105	659,700	1	43	65.2
		1106	396,000	1	11	27.8
	120	1201	492,300	1	22	44.7
		1202	312,300	1	7	22.4
		1203	710,100	1	19	26.8	44.2	±	17.6
		1204	321,300	1	14	43.6
		1205	506,700	1	33	65.1
		1206	495,900	1	31	62.5
	400	1301	488,700	1	30	61.4
		1302	314,100	1	10	31.8
		1303	396,000	1	22	55.6	58.0	±	31.5
		1304	676,800	1	80	118.2
		1305	459,900	1	20	43.5
		1306	564,300	1	21	37.2
ENU	100	1401	501,300	1	92	183.5
		1402	729,000	1	101	138.5
		1403	556,200	1	84	151.0	161.3	±	17.1	*(S)
		1404	324,900	1	58	178.5
		1405	530,100	1	86	162.2
		1406	343,800	1	53	154.2

D.W.: Negative control (Water for injection, 10 mL/kg)

ENU: Positive control (N -ethyl-N -nitrosourea, 10 mL/kg, i.p., dose once a day, for 2 days, expression period; 10 days)

*: Significant difference from negative control (p<0.05), (S): Student's t test

 

Table 5: Induction of mutation in stomach of transgenic mice treated with the test item (Additional assay) [Male mice dosed once a day, for 28 days (Oral administration, 3 days after final administration)]

Substance	Dose (mg/kg bw/day, p.o.)	Animal ID No.	Number of plaque forming units	Number of packagings	Number of mutants	Mutant frequency (×10^-^6)	Group Mean ± S.D. (×10^-^6)
D.W.	0	1001	445,500	1	27	60.6
		1002	322,200	1	11	34.1
		1003	312,300	1	13	41.6	47.7	±	11.8
		1004	346,500	1	15	43.3
		1005	437,400	1	28	64.0
		1006	330,300	1	14	42.4
Test item	40.0	1101	326,700	1	22	67.3
		1102	740,700	1	36	48.6
		1103	517,500	1	35	67.6	52.7	±	12.6
		1104	317,700	1	15	47.2
		1105	342,000	1	12	35.1
		1106	356,400	1	18	50.5
	120	1201	554,400	1	26	46.9
		1202	309,600	1	12	38.8
		1203	513,000	1	34	66.3	47.0	±	10.4
		1204	405,900	1	15	37.0
		1205	518,400	1	24	46.3
		1206	362,700	1	17	46.9
	400	1301	400,500	1	16	40.0
		1302	482,400	1	30	62.2
		1303	389,700	1	22	56.5	52.8	±	11.6
		1304	482,400	1	32	66.3
		1305	424,800	1	16	37.7
		1306	332,100	1	18	54.2
ENU	100	1401	313,200	1	110	351.2
		1402	323,100	1	131	405.4
		1403	513,900	1	235	457.3	412.1	±	98.6	*(AW)
		1404	353,700	1	119	336.4
		1405	474,300	1	279	588.2
		1406	323,100	1	108	334.3

D.W.: Negative control (Water for injection, 10 mL/kg)

ENU: Positive control (N -ethyl-N -nitrosourea, 10 mL/kg, i.p., dose once a day, for 2 days, expression period; 10 days)

*: Significant difference from negative control (p<0.05), (AW): Aspin-Welch's t test

 

Table 6: Histopathological findings in the gene mutation assay [Male mice dosed once a day, for 28 days (Oral administration, 3 days after final administration)]

Substance		D.W.	Test item (mg/kg bw/day, p.o.)
Organ	Findings	0	40.0	120	400
Liver		(7)	(7)	(7)	(7)
	no remarkable change	7	7	7	0
	eosinophilic change, slight	0	0	0	7
Duodenum		(7)	(7)	(7)	(7)
	no remarkable change	7	7	7	7
Forestomach		(7)	(7)	(7)	(7)
	no remarkable change	7	7	7	1
	erosion/ulcer, slight	0	0	0	1
	erosion/ulcer, moderate	0	0	0	5
	hyperkeratosis, slight	0	0	0	6
	hyperplasia, squamous cell, slight	0	0	0	6
Glandular stomach		(7)	(7)	(7)	(7)
	no remarkable change	7	7	6	5
	infiltration, inflammatory cell, focal, slight	0	0	1	2

D.W.: Negative control (Water for injection, 10 mL/kg)

(): Number of animals examined microscopically at this site

 

Table 7: True mutations and their frequencies identified in the 30 mutant plaques analyzed from the liver tissue of animal 1103 (low dose group).

Sample	Position	Mutation	Mutation frequency	Mutation count
Liver 1103 – PCR 1	38	1 bp deletion (C)	0.05691	1.7
Liver 1103 – PCR 2			0.05098	1.5
Liver 1103 – PCR 1	1090	G:C -> A:T	0.05367	1.6
Liver 1103 – PCR 2			0.08979	2.7
Liver 1103 – PCR 1	1627	G:C -> A:T	0.70279	21.1
Liver 1103 – PCR 2			0.64446	19.3
Liver 1103 – PCR 1	2813	G:C -> C:G	0.07142	2.1
Liver 1103 – PCR 2			0.09843	3.0

 

Table 8: True mutations and their frequencies identified in the 30 mutant plaques analyzed from the liver of animal 1304 (high dose group).

Sample	Position	Mutation	Mutation frequency	Mutation count
Liver 1103 – PCR 1	1090	G:C -> T:A	0.05407	1.6
Liver 1103 – PCR 2			0.05680	1.7
Liver 1103 – PCR 1	1187	G:C -> A:T	0.89764	26.9
Liver 1103 – PCR 2			0.90901	27.3

Conclusions:

The test substance did not induce gene mutation in either the liver, stomach or duodenum of transgenic mice under the conditions in this study.

Executive summary:

A gene mutation assay according to OECD TG 488 and GLP with transgenic mice (MutaMouse) was conducted to assess the potential of the test substance to induce gene mutation (reporter gene: lacZ) in the liver, stomach and duodenum in vivo.

The test substance was administered to male transgenic mice orally, once a day, for 28 consecutive days by gavage at the dose levels of 40.0, 120, and 400 mg/kg bw/day. After 3 days of manifestation period, the mutant frequencies in the liver, stomach and duodenum were determined.
The negative control values obtained for all organs were within the acceptable range of the historical control data and thus considered as valid. The mean of the mutant frequencies in the liver, stomach and duodenum of the animals treated with the test substance did not show any increases as compared to the concurrent negative control. However, in some samples of the liver and the stomach the individual values surpassed higher the upper value of the historical control range. Therefore,  the liver and stomach were re-assayed from genomic DNA extraction for all animals.

All individual mutation frequencies in stomach were within the historical data in the additional assay. In the liver, the mutation frequencies still exceed the historical control range for two animals. A sequence analysis of mutant plaques from these two animals was performed to confirm, if the increased mutant frequencies were induced by a true mutagenic effect of the test substance. The results of the sequencing (Next generation sequencing) indicated, that the increased mutant frequencies observed in the liver are likely a result from clonal expansion of single mutations. It was concluded that it do not represent a true mutagenic effect of the test substance.

In the positive control group, treated with N-ethyl-N-nitrosourea (ENU), the mutant frequencies in the liver, stomach and duodenum were statistically significant increased compared with the negative control group. Therefore, the present study was judged to be properly conducted.

Considering all information available, including statistical analysis, it was concluded that the test substance did not induce gene mutation in either the liver, stomach or duodenum of transgenic mice (negative) under the conditions in this study.

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2001-09-12 to 2001-11-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1997)

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B 61
- Expiration date of the lot/batch:
- Purity test date: October 31, 2001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Glyoxal as 40% aqueous solution is stable for 6 months. The stability of the test substance as such and throughout the study period had been verified by reanalysis
- Solubility and stability of the test substance in the solvent/vehicle: the stability of the test substance (aqueous solution) at room temperature in the vehicle water also had been determined analytically in the course of the determination of the reanalysis

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate, UK
- Age and weight at study initiation:
Range-finding study: the males were about 7 weeks old whereas the females were about 8 to 9 weeks old. At test initiation the males weighed between 189 and 196 g and the females weighed between 152 and 179 g.
Main test: only males were used, they were about 7 weeks old and weighed between 196 and 231 g at test initiation.
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: housed in groups of no more than four animals in solid-floored cages
- Diet: special diet, ad libitum
- Water: public supply water, ad libitum
- Acclimation period:
Range-finding study: 1 to 7 days
Main test: 7 to 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 47 - 67
- Air changes (per hr): at least 15 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

OTHER
in order to enrich the environment and enhance the welfare of the animals, they were provided with wooden Aspen chew blocks .

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PRELIMINARY RANGE FINDER
A group of 3 male rats were dosed once with 2540 mg/kg bw Glyoxal and 2 groups of three female rats were dosed once with 1270 mg/kg bw or 2540 mg/kg bw of the test substance. Dosing consisted of a single application by gavage. The animals were observed for clinical toxicity, and on the basis of the results a maximum tolerable dose was selected for the main test.

MAIN ASSAY
- On the basis of the results of the range finding test, 2000 mg/kg bw was selected as maximum tolerable dose for male Wistar rats;
- The assay comprised two experiments with different post-treatment period: 12 –14 h and 2 – 4 h;
- The animals received single oral application of test solution at an application volume of 10 mL/kg bw;
- The test animals were examined for clinical signs of toxicity and the body weights were recorded;
- At the end of the respective post-treatment periods, the rats were sacrificed and the hepatocytes were isolated from the liver of each animal.

Duration of treatment / exposure:

Not applicable as single application

Frequency of treatment:

single administration by gavage

Post exposure period:

2-4 h or 12-14 h

Dose / conc.:

1 000 other: mg/kg bw

Remarks:

Main assay; actual ingested
(the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

Dose / conc.:

2 000 other: mg/kg bw

Remarks:

Main assay; actual ingested
(the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

Dose / conc.:

1 270 other: mg/kg bw

Remarks:

Range-Finding test; actual ingested
(the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

Dose / conc.:

2 540 other: mg/kg bw

Remarks:

Range-Finding test; actual ingested
(the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

No. of animals per sex per dose:

RANGE-FINDING
A group of 3 male rats were dosed once with 2540 mg/kg bw Glyoxal and 2 groups of three female rats were dosed once with 1270 mg/kg bw or 2540 mg/kg bw of the test substance.

MAIN TEST
A total of 32 males were used; each group comprised 4 animals.

Control animals:

yes

Positive control(s):

2-Acetamidofluorene (2-AAF, Sigma Chemical Co. Poole, UK) in corn oil, 75 mg/kg bw, was used for experiment 1.
Dimethylnitrosamine (DMN, Sigma Chemical Co. Poole, UK) in purified water, 10 mg/kg bw, was used for experiment 2.

Tissues and cell types examined:

- At the end of the respective post-treatment periods, the rats were sacrificed and the hepatocytes were isolated from the liver of each rat;
- The cells were examined for cell viability as measured by the Trypan blue exclusion technique;
- DNA damage and repair was measured by incorporation of 3H-thymidine using autoradiography technique;
- For evaluation and quantification of UDS, a total of 100 cells/animal was examined and following parameters were considered: net nuclear grain (NNG) count/cell, group mean net nuclear grain (NNG) count, mean net grain (NG) count of cells in repair, percentage of cells in repair (cells with NNG >= 5).

Evaluation criteria:

- A positive response implicates a dose-related increase in mean number of NNG counts (> 0 at one of the test points) and in percentage of cells in repair (i.e. cells with NNG >= 5), which must be >= 20%.
- A negative response implicates that both, the NNG counts and the percentage of cells in repair are within the range of negative control.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Glyoxal at 1000 and 2000 mg/kg bw resulted in NNG values < 0. In fact, group mean NNG values for the two experiments were in the range -0.8 to -2.6, and no more than 4.0% cells were seen in repair at any of the tested doses.

Toxicity:

no effects

Remarks:

In the main study one animal was found dead (first experiment, 2000 mg/kg dose level), which was surprising and not consistent with results of the range-finder experiment. No other clinical signs of toxicity were observed in any other main study animal.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
During the 2 day post-exposure period, the males treated with 2540 mg/kg bw showed weight loss whereas the females treated with the same dose were cold to the touch, pale, lethargic, with hunched posture and weight loss. One female animal was sacrificed in extremis whereas another one was found dead. No mortalities were seen in the group of females treated with 1270 mg/kg bw; the animals displayed piloerection, lethargy, abnormal breathing, protruding eyes and weight loss.

RESULTS OF DEFINITIVE STUDY
Cell viability in the first experiment at both dose levels (1000 and 2000 mg/kg bw) was quite similar and ranged between 55 and 67%; cell viability in both cases was within or close to negative (vehicle) control range (59 – 66%). Cell viability in the positive control group treated with 2-AAF also was within negative control range (55 – 67%).
Cell viability in the second experiment at both dose levels (1000 and 2000 mg/kg bw) was quite similar and ranged between 63 and 74%; cell viability in both cases was within or close to negative (vehicle) control range (69 – 78%). Cell viability in the positive control group treated with DMN also was within negative control range (70 – 77%).
For details on genotoxicity, see tables below:

Experiment 1, summary of findings referring to the group mean net grain count:

12 to 14 hour sacrifice time
Group	Dose (mg/kg bw)	Net grain count (NNG)		% of cells in repair (NNG ≥ 5)		Net grain count of cells in repair
		Mean	SD	Mean	SD	Mean	SD
Neg. control	0	- 2.6	0.3	-	-	-	-
Glyoxal	1000	- 2.3	0.8	-	-	-	-
	2000	- 2.6	0.8	-	-	-	-
2-AAF	75	26.5	11.5	99.0	1.0	26.7	11.3

Experiment 2, summary offindings referring to the group mean net grain count:

2 to 4 hour sacrifice time
Group	Dose (mg/kg bw)	Net grain count (NNG)		% of cells in repair (NNG ≥ 5)		Net grain count of cells in repair
		Mean	SD	Mean	SD	Mean	SD
Neg. control	0	- 0.8	1.1	3.3	2.3	6.0	0.6
Glyoxal	1000	- 0.8	1.3	4.0	4.0	6.5	0.3
	2000	- 1.3	1.5	3.3	2.1	6.1	0.7
DMN	10	25.4	1.4	92.7	2.5	27.2	1.7

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

It was reported that two slides, which clearly were from different animals, had been erroneously coded with the same label, and it was not possible to assign the slides to the animals. Therefore, the data obtained from these two slides were excluded from data analysis. As data from at least five animals were available for each test group, this exclusion of data did not affect the validity of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1997)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

(1998)

Qualifier:

according to guideline

Guideline:

other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests (1995)

GLP compliance:

yes

Type of assay:

other: micronucleus assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B 61
- Purity test date: October 31, 2001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Glyoxal as 40% aqueous solution is stable for 6 months. The stability of the test substance as such and throughout the study period had been verified by reanalysis
- Solubility and stability of the test substance in the solvent/vehicle: the stability of the test substance (aqueous solution) at room temperature in the vehicle water also had been determined analytically in the course of the determination of the reanalysis

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate, UK
- Age and weight at study initiation:
Range-finding study: animals of both sexes were used; at test initiation the mice were about 5 to 7 weeks old and their weights ranged from 18 to 34 g.
Main test: only male animals were used; at test initiation the mice were about 5 to 6 weeks old and their weights ranged from 25 to 32 g.
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: housed in groups of no more than 3 animals of the same sex in appropriate caging (in the range-finding study, some of the animals were housed individually during the experimental phase)
- Diet (e.g. ad libitum): special diet, ad libitum
- Water (e.g. ad libitum): public supply water, ad libitum
- Acclimation period:
Range-finding study: 1 to 9 days
Main test: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 47 - 61
- Air changes (per hr): at least 15 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

OTHER
in order to enrich the environment and enhance the welfare of the animals, they were provided with wooden Aspen chew blocks

Route of administration:

intraperitoneal

Vehicle:

water

Details on exposure:

PRELIMINARY RANGE FINDER
A preliminary range-finding test was conducted for assessment of the maximum tolerable dose for the main assay; the application volume was 10 mL/kg bw. As the findings of the preliminary test revealed no differences between males and females, it was decided to use only male mice for the main test.
MAIN ASSAY
- According to the results of the preliminary test, 225 mg/kg bw was selected as maximum tolerable dose for the main assay, and the remaining test doses were 56.25 and 112.5 mg/kg bw;
- Each dose group of the main test comprised 6 animals (males);
- Each animal received 2 i.p. applications on two consecutive days (i.e. at 24 hours interval);
- Following dosage, the animals were observed for overt signs of toxicity and mortality;
- At the end of a post treatment period of 24 hours, the animals were sacrificed and the femoral bone marrow was extracted.

Duration of treatment / exposure:

2 days

Frequency of treatment:

2 i.p. applications at 24 hours interval

Post exposure period:

24 hours

Dose / conc.:

127 mg/kg bw (total dose)

Remarks:

Range-Finding test; injected i.p. (the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

Dose / conc.:

178 mg/kg bw (total dose)

Remarks:

Range-Finding test; injected i.p. (the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

Dose / conc.:

225 mg/kg bw (total dose)

Remarks:

Range-Finding test; injected i.p. (the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

Dose / conc.:

320 mg/kg bw (total dose)

Remarks:

Range-Finding test; injected i.p. (the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

Dose / conc.:

635 mg/kg bw (total dose)

Remarks:

Range-Finding test; injected i.p. (the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

Dose / conc.:

2 540 mg/kg bw (total dose)

Remarks:

Range-Finding test; injected i.p. (the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

Dose / conc.:

56.25 mg/kg bw/day (actual dose received)

Remarks:

Main test; injected i.p. (the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

Dose / conc.:

112.5 mg/kg bw/day (actual dose received)

Remarks:

Main test; injected i.p. (the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

Dose / conc.:

225 mg/kg bw/day (actual dose received)

Remarks:

Main test; injected i.p. (the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)

No. of animals per sex per dose:

RANGE-FINDING
- The two lowest dose groups consisted of 2 animals (1 male and 1 female) each;
- The 225 mg/kg bw group consisted of 4 animals (3 males and 1 female);
- Each of the 320 and the 635 mg/kg bw groups consisted of 2 animals (1 male and 1 female);
- The 2540 mg/kg bw group consisted of 6 animals (3/sex).

MAIN TEST
Each dose group of the main test comprised 6 males.

Control animals:

yes

Positive control(s):

Cyclophosphamide (CPA, 4 mg/mL in physiological saline) was used as positive control substance was administered as single i.p. application at a dose of 40 mg/kg bw.

Tissues and cell types examined:

- The femoral bone marrow was extracted, prepared and examined for the polychromatic/normochromatic erythrocytes ratio (PCE/NCE ratio) and the number of micronuclei in 1000 polychromatic erythrocytes per animal;
- The mean group frequencies of micronucleated PCE in vehicle control were compared with the historical negative control range; the assay was valid when the incidence of micronucleated PCE in the vehicle control was within/close to the historical vehicle control range as reported in the table below (laboratory data, 2000). Furthermore, the assay was valid if at least 5 animals/ per group were available for analysis, and if the positive control chemical (CPA) induced a statistically significant increase in the frequency of micronucleated PCE.

Evaluation criteria:

The test substance was considered as positive if a statistically significant increase in the frequency of micronucleated PCE was seen in at least one dose, and if the frequency of micronucleated PCE exceeded the historical vehicle control range.

Statistics:

- The individual and the group mean frequency of micronucleated PCE/1000 cells (± standard deviation) were determined;
- For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity Chi2 test;
- The numbers of micronucleated PCE in each treated group were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine Chi2; probability values of p ≤ 0.05 were accepted as significant.
- A further statistical test (for linear trend) was used to evaluate possible dose-response relationships;
- When the heterogeneity Chi2 test provided evidence of significant variability between animals within at least one group (p ≤ 0.05), non-parametric analysis was used (Wilcoxon rank sum).

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

All treated groups exhibited PCE/NCE ratios similar to or slightly lower than in the vehicle control. Group mean frequencies of micronucleated PCE from treated animals were also similar to or lower than the value in the vehicle control.

Toxicity:

yes

Remarks:

Lethargy was the only clinical effect reported and was seen in the high dose group (225 mg/kg bw/d); no mortality was reported. At this dose a slight decrease in PCE/NCE ratio observed in comparison to vehicle control indicated bone marrow toxicity.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

PCE/NCE ratio, summary of group mean data:

Treatment group	Mean ratio PCE/NCE
Vehicle control	0.95
Glyoxal 56.25 mg/kg bw/day	0.98
Glyoxal 112.5 mg/kg bw/day	0.89
Glyoxal 225 mg/kg bw/day	0.71
Positive control (CPA, 40 mg/kg bw)	1.17

Frequencies of micronucleated PCE, summary of group mean data:

Treatment group	Group mean frequency of micronucleated PCE/1000 cells per treatment group (± SD)
Vehicle control	0.67±0.41
Glyoxal 56.25 mg/kg bw/day	0.70±0.57
Glyoxal 112.5 mg/kg bw/day	0.67±0.41
Glyoxal 225 mg/kg bw/day	0.25±0.27
Positive control (CPA, 40 mg/kg bw)	13.10±6.27**
**, p ≤ 0.001

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Testing of Glyoxal (40% aqueous solution) in the standard plate incorporation test method based on Ames et al. (Mut. Res., 31, 347-364, 1975 and Proc. Nat. Acad. Sci. USA, 70, 782-786, 1973) and according to OECD TG 471, revealed an increase in the number of his+ revertants for the Salmonella typhimurium strains TA 100 and TA 102, both with and without S9 mix (BASF 40M0496/014142). The remaining strains (TA 1535, TA 1537 and TA 98) were inconspicuous in the presence and absence of S9 mix. The results of the supporting study (Pharma Research Toxicology 84.0205) are in accordance with those mentioned above, and furthermore, positive results were also shown for E. coli WP2 uvrA. In six further Ames tests Glyoxal was mutagenic under the experimental conditions chosen (Kato F 1989, Jetoc 1997; Societe Francaise Hoechst 82/0790; Hoechst 880271; Jetoc 2008; Jung et al 1992).

Glyoxal (40% aqueous solution) was further tested for clastogenicity in the in vitro mammalian chromosome aberration test according to OECD TG 473, using cultured Chinese hamster ovary (CHO) cells (BASF SE 729/211-D6172). In the presence of S-9 mix, treatment of CHO cells with 371.2 and 580 μg/mL Glyoxal resulted in statistically significant increases in numbers of cells with structural aberrations as compared to concurrent vehicle controls. In the absence of S-9 mix, treatment of CHO cells with 580 μg/mL Glyoxal resulted in a statistically significant increase in the numbers of cells with structural aberrations as compared to concurrent vehicle controls. Structural chromosomal aberrations consisted of chromosome deletions, chromatid deletions, and chromatid exchanges.

In two supporting studies Glyoxal 40% induced chromosome aberrations in cultured Chinese hamster ovary cells (Henkel 0367/EC 124) and Chinese hamster lung fibroblasts (Jetoc 1997). In a sister chromatid exchange assay according OECD TG 479 Glyoxal 40% induced sister chromatid exchange in CHO cells under the test conditions used (Pharmakon Research CT-096-82).

The mutagenic potential of Glyoxal (40% aqueous solution) was also assessed in the in vitro mammalian cell forward gene mutation assay according to OECD TG 476 using L5178Y mouse lymphoma cells (BASF SE 729/213-D6173). In the first experiment, no statistically significant increases in mutant frequency were observed at any concentration level, both in the absence and the presence of S-9 mix. In contrast, in the second experiment, in the absence of S-9, statistically significant increases in mutant frequency were observed at the highest two doses analyzed (250 and 300 μg/mL); a statistically highly significant dose-related linear trend was also reported. In the presence of S-9, no such increase in mutant frequency could be observed. The third, confirmatory experiment confirmed the increase in mutant frequencies in the absence of S-9 mix, at Glyoxal concentrations of 275, 325 and 375 μg/mL; moreover, in this experiment increased mutant frequencies compared to the concurrent solvent controls were observed throughout the dose range, and a statistically highly significant dose-related linear trend was evident. In a further no-guideline in vitro mammalian cell transformation assay no genotoxicity was detected in Chinese hamster lung fibroblasts (CIT 87.0418). In a DNA repair test Glyoxal 40% induced unscheduled DNA repair in primary rat hepatocytes (Pharmakon Research PH-311-AC-004-82).

 

In vivo

The clastogenic potential of Glyoxal was evaluated in the in vivo micronucleus assay with mouse bone marrow according to OECD TG 474, using CD1 out-bred mice which received two consecutive i.p. injections of the test substance (BASF SE 729/215-D6172). Referring to clastogenicity, all treated groups exhibited PCE/NCE ratios that were similar to or slightly lower than the vehicle controls. Moreover, in the treated groups, as the dose level increased there was a slight decrease in PCE/NCE ratio when compared to the vehicle control group; this was considered to be a possible indication of substance-related bone marrow toxicity. Group mean frequencies of micronucleated PCE from animals treated with Glyoxal were also similar to or lower than the value in the vehicle control group; no statistically significant differences were evident. Thus, Glyoxal did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated by i.p. at up to 225 mg/kg/day, a dose level shown to cause clinical signs and indications of bone marrow toxicity. The findings of two further MNT studies were in accordance with the results above since in the first supporting study (Société Francaise Hoechst 2018 MAS), Glyoxal did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated by oral gavage with 1000 mg/kg/day of test substance; in the second study (Pharmakon PH 309A-AC-002-82), findings were similar since Glyoxal did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated i.p. with 400 mg/kg/day; a dose level shown to cause clinical toxicity.

The genotoxic potential of Glyoxal was also assessed in the in vivo unscheduled DNA synthesis assay with hepatocytes isolated from male Wistar rats following single oral dosage by gavage; the test was conducted according to OECD TG 486 (BASF SE 729/217-D6173). The assay comprised two experiments with different post-treatment periods of 12 to 14 and 2 to 4 hours, respectively. At the end of the post-treatment period, the rats were sacrificed and the hepatocytes were isolated from the livers. The cells were examined for cell viability, and DNA damage and repair was measured by incorporation of 3H-thymidine using autoradiography technique. A single case of mortality was reported at 2000 mg/kg bw which probably was incidental. Referring to genotoxicity, oral treatment with Glyoxal at 1000 and 2000 mg/kg bw resulted in net nuclear grain count values < 0. In fact, group mean NNG values for the two experiments were in the range -0.8 to -2.6, and no more than 4.0% cells were in repair at any of the doses tested, indicating that single oral dosage of male rats with 1000 or 2000 mg/kg bw of Glyoxal did not result in increased UDS in hepatocytes isolated approximately 12-14 or 2-4 hours after dosing. In an in vivo UDS test with oral application of Glyoxal to rats, unscheduled DNA synthesis was detected in the pyloric mucosa (Furihata 1985). An induction of an up to 100 -fold increase in ornithine decarboxylase activity and a more than 10 -fold increase in DNA synthesis were reported; furthermore, apparent unscheduled DNA synthesis in the pyloric mucosa of the stomach could be evidenced within 3 hours following dosage. These results suggest that Glyoxal has a potential tumour-promoting activity and may also initiate carcinogenesis in the glandular stomach of rats. The genotoxicity of glyoxal in the glandular stomach of F344 rats was confirmed by the alkaline elution method (Furihata, 1989). Glyoxal at doses of 50-550 mg/kg bw could be shown to induce DNA damage in the pyloric mucosa of rat stomach. Thus Glyoxal was confirmed to be genotoxic on this tissue. Ueno et al. (1991b) investigated the potential of Glyoxal to induce DNA damage in form of single strand breaks in various organs of Sprague-Dawley rats by the alkaline elution technique. DNA lesions were detected in the liver at all dose levels. The alkaline elution of DNA from other tissues (kidney, spleen, pancreas, and lung) following oral exposure of rats to 1000 mg/kg bw showed no biologically relevant DNA lesions. The genotoxicity of Glyoxal in the stomach of rat and the demonstrated potential of Glyoxal to induce DNA damage in form of single strand breaks in the liver, indicate that Glyoxal reacts at the point of entry, namely the stomach and immediately downstream in the liver but only at high dose levels as indication that the detoxifying capacity is overloaded. However, at low dose levels with sufficiently acting enzymatic activity in the liver or in distant organs or tissues no effects were noted. In a further study Glyoxal 40% did not induce unscheduled DNA synthesis in primary hepatocytes of male Wistar rats treated by oral gavage (CCR 230602).

Furthermore, there were no indications of a mutagenic potential in germ cells in the two sex-linked recessive lethal (SLRL) tests using Drosophila melanogaster (Barnett BM 1989 and American Cyanamid Company 1983).

A gene mutation assay according to OECD TG 488 and GLP with transgenic mice (MutaMouse) was conducted to assess the potential of the test substance to induce gene mutation (reporter gene: lacZ) in the liver, stomach and duodenum in vivo (BASF SE 60M0496/01X123). The test substance was administered to male transgenic mice orally, once a day, for 28 consecutive days by gavage at the dose levels of 40.0, 120, and 400 mg/kg bw/day. After 3 days of manifestation period, the mutant frequencies in the liver, stomach and duodenum were determined.
The negative control values obtained for all organs were within the acceptable range of the historical control data and thus considered as valid. The mean of the mutant frequencies in the liver, stomach and duodenum of the animals treated with the test substance did not show any increases as compared to the concurrent negative control. However, in some samples of the liver and the stomach the individual values surpassed higher the upper value of the historical control range. Therefore,  the liver and stomach were re-assayed from genomic DNA extraction for all animals. All individual mutation frequencies in stomach were within the historical data in the additional assay. In the liver, the mutation frequencies still exceed the historical control range for two animals. A sequence analysis of mutant plaques from these two animals was performed to confirm, if the increased mutant frequencies were induced by a true mutagenic effect of the test substance. The results of the sequencing (Next generation sequencing) indicated, that the increased mutant frequencies observed in the liver are likely a result from clonal expansion of single mutations. It was concluded that it do not represent a true mutagenic effect of the test substance. In the positive control group, treated with N-ethyl-N-nitrosourea (ENU), the mutant frequencies in the liver, stomach and duodenum were statistically significant increased compared with the negative control group. Therefore, the present study was judged to be properly conducted. Considering all information available, including statistical analysis, it was concluded that the test substance did not induce gene mutation in either the liver, stomach or duodenum of transgenic mice (negative) under the conditions in this study.

 

Summary

In vitro, Glyoxal was shown to be mutagenic in bacteria and to induce an increase in structural chromosome aberrations in CHO, both, in the absence and presence of S9 mix. Glyoxal further showed some evidence of mutagenic activity at the hprt locus (6-thioguanine resistance) of L5178Y mouse lymphoma cells in the absence of metabolic activation but not in the presence of S9 mix, when investigated up to its limit of cytotoxicity.

In vivo, Glyoxal showed positive results, which are restricted to the pyloric mucosa of the stomach and to the liver and were induced at high dose levels of Glyoxal (50 mg/kg bw) and unknown purity. However, no clastogenic potential could be evidenced for pure Glyoxal in modern guideline studies according to GLP, such as the micronucleus test, and the UDS assay which revealed no genotoxic potential. In a recent somatic cell gene mutation assay in transgenic mice (TGR) the test substance did not induce gene mutation in either the liver, stomach or duodenum. All in all, there is no mutagenic potential of Glyoxal in vivo germ cells.

Glyoxal is as a dialdeyhde very reactive, which explains the positive in vitro results. However, those effects were not reproducible with pure Glyoxal in modern in vivo guideline studies according to GLP.

 

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 the purpose of the assessment of a potential classification under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776. Based on all available data the registered substance should not be subject to classification for mutagenicity.

However, as the substance is already listed in Annex VI of Regulation (EC) No 1272/2008, a classification as mutagenic cat.2 (H341:"Suspected of causing genetic defects") in the EU is required.