Glutaral

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Glutaraldehyde is mutagenic to bacteria with and without metabolic activation in an Ames test conducted according to guideline and GLP.

Glutaraldehyde is clastogenic in mammalian cells with and without metabolic activation in an In Vitro Mammalian Chromosome Aberration Test according to OECD TG 473 and GLP.

Glutaraldehyde is mutagenic in mammalian cells in an in vitro mammalian cell gene mutation test using the xprt gene conducted according to guideline and GLP.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

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)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Physical state: colourless liquid
- Analytical purity: ca. 50% (analytical report data)
- Impurities (identity and concentrations): methanol 0.2%
- Composition of test material, percentage of components: 50.3% glutaraldehyde, 46% water, 0.2% methanol
- Lot/batch No.: 50-4402
- Stability under test conditions: the stability of the test substance at room temperature in water over a period of 14 days had been verified analytically (Bioanalytical Laboratory of the Experimental Toxicology and Ecology, BASF AG)
- Storage condition of test material: refrigerator, under N2 conditions

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix was prepared according to Ames et al. (Mut. Res. 31:347-364, 1975) from the S9 liver fraction of Aroclor 1254-treated male Sprague-Dawley rats, mixed with appropriate cofactors.

Test concentrations with justification for top dose:

A range-finding cytotoxicity test was performed prior to the main test the test concentrations for the main assay were selected on the basis of the results of the pre-test:
Without S9 mix: 0.25, 0.5 and 1 µg/mL
With S9 mix: 2.5, 5 and 10 µg/mL

Vehicle / solvent:

The cell culture medium MEM medium with Earle´s salt was used as vehicle.

Untreated negative controls:

yes

Remarks:

the untreated negative controls were performed with and without S9 mix and consisted of cells in culture medium (MEM medium with Earle´s salt).

Negative solvent / vehicle controls:

yes

Remarks:

the vehicle control was identical to the untreated control.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

CYTOTOXICITY
A range-finding cytotoxicity test was performed prior to the main test. The test concentrations in the absence of S9 mix were 0.25, 0.5, 1.0, 2.0 and 4.0 µg/mL; the test concentrations in presence of S9 mix were 1.25, 2.5, 5.0, 10 and 20 µg/mL.

MAIN ASSAY
The test substance was dissolved in 1mL serum-free culture medium and was then added to the culture medium with or without 1 mL of S9 mix.
The cells were incubated in the test solutions over a period of 4 hours.
Following exposure of the cells to the test material, the culture medium was replaced by fresh medium supplemented with 10% fetal calf serum and the cells were incubated again for further 14 hours until harvesting. Approximatively 2 to 3 hours prior harvesting, 0.2 µg/mL Colcemid in culture medium was added to the cells in order to stop mitosis in the metaphase. The cells were harvested after a total period of 18 hours, corresponding to 1 - 1.5 x the normal cycle time of the cells.
For harvesting, the culture medium was completely removed and the cells were subjected to an hypotonic treatment (0.4% KCl, 37 °C, 20 minutes); thereafter they were fixed (methanol: glacial acetic acid, 3:1), dried and stained on the slides with a solution of Giemsa and Titrisol. The cell preparations were mounted using Corbit-Balsam.
In each culture, 200 (100 for positive controls) well-spread metaphases were counted and cells displaying 20 to 22 chromosomes were examined for structural and numerical chromosome aberrations.

Evaluation criteria:

- Cells displaying 20 to 22 chromosomes were examined for structural chromosome aberrations, such as gaps, breaks, fragments, segment loss, multiple aberrations, pulverization (break down of chromosomes into irregular particles) and/or exchanges.
- Numerical aberrations such as e.g. hyperploidy (metaphase with additional chromosomes) also were considered.
- The mitotic index was determined and the cells were counted for determination of cytotoxicity using additional cultures treated as those of the main test.
The definitions used within the chromosomal analysis were based on following references:
(1)- Evans HJ, O'Riordan ML (1975) Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutat Res. 31(3): 135-48 (published)
(2)- Savage JRK (1975) Classification and relationships of induced chromosomal structural changes. J. Med. Genet. 12: 103-122 (published)
(3)- Standard-Protokoll zur cytogenetischen Auswertung von Mitose- und Meiose-chromosomen bei der Routineuntersuchung; ausgearbeitet von der Arbeitsgruppe der Industrie, Cytogenetik“, 1987.

Statistics:

The statistical evaluation of the findings was carried out using the MUCHAN program system (BASF AG). Fisher´s exact test for the hypothesis of equal proportions was used for the comparison of each test dose with the control. The test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Glutaraldehdyde caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases (with/without gaps).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cell growth inhibition indicative of cytotoxicity was observed from 1 µg/mL in the absence of S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Glutaraldehyde caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases (with/without gaps).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cell growth inhibition indicative of cytotoxicity was observed at the highest tested concentration of 20 µg/mL in the presence of S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Mitotic index (based on 1000 cells/culture):
A weak suppression of the mitotic activity was observed under all experimental conditions, as shown in following table 3.

Cell attachment to the slides and quality of the metaphases:
The treatment of the cells with the different doses of test substance did not disturb cell attachment on the slides. Within the dose-range that was tested, the quality of the metaphases was sufficiently good to allow chromosomal evaluation.

Cytotoxicity, cell count:
A slight growth inhibition of the cells was observed under all experimental conditions, as shown in table 4.

Table 1. Glutaraldehyde-induced chromosome aberration, without metabolic activation:

Dose (µg/ml)	Metaphases with aberrations^a
	Incl. Gaps (%)	Excl. Gaps (%)	EX (%)	MA (%)	CD (%)	AP (%)	PP (%)	EP (%)
Neg. Cont.	3.0	2.0	1.0	0.0	0.0	0.0	0.5	0.0
0.25	4.0	4.0	2.5	0.0	0.0	0.0	0.0	0.0
0.50	5.0	4.0	3.0	0.0	0.0	0.0	0.5	0.0
1.00	12**	11**	11**	0.0	0.0	0.0	0.0	0.0
Pos. Cont.(EMS)	19**	19**	12**	0.0	0.0	0.0	0.0	0.0

^a, Total number of metaphases: 200 (except for positive control, 100)

EX, exchanges

MA, multiple aberrations

CD, chromosomal disintegration

AP, aneuploidy

PP, polyploidy

EP, endoploidy

*: p<=0.05; **: p<=0.01

Table 2. Glutaraldehyde-induced chromosome aberration, with metabolic activation:

Dose (µg/ml)	Metaphases with aberrations^a
	Incl. Gaps (%)	Excl. Gaps (%)	EX (%)	MA (%)	CD (%)	AP (%)	PP (%)	EP (%)
Negative Control	3.0	1.5	0.0	0.0	0.0	0.0	0.0	0.0
2.50	3.5	2.5	2.5*	0.0	0.0	0.0	0.0	1.0
5.00	6.5	6.5*	5.0**	0.0	0.0	0.0	0.5	0.5
10.00	8.0	7.5**	6.5**	0.0	0.0	0.0	0.0	0.0
Positive Control (CPP)	19**	17**	12**	0.0	0.0	0.0	0.0	0.0

^a, Total number of metaphases: 200 (except for positive control, 100)

EX, exchanges

MA, multiple aberrations

CD, chromosomal disintegration

AP, aneuploidy

PP, polyploidy

EP, endoploidy

*: p<=0.05; **: p<=0.01

Table 3. Mitotic index data:

Without S9 mix
Test groups	Relative percentage of mitotic cells (i.e. versus 100% for negative control)
Negative control	100%
0.25 µg/ml	69.2%
0.50 µg/ml	77.1%
1.00 µg/ml	71.8%
Positive control ()	74.0%
With S9 mix
Negative control	100%
2.50 µg/ml	65.9%
5.00 µg/ml	75%
10.0 µg/ml	54.1%
Positive control (CPP)	56.3%

Table 4. Cytotoxicity, cell count data:

Without S9 mix
Test groups	Cell count as percentage of negative control
Negative control	100%
0.25 µg/ml	106.6%
0.50 µg/ml	107.6%
1.00 µg/ml	64.0%
2.00 µg/ml	73.1%
4.00 µg/ml	57.9%
With S9 mix
Negative control	100%
1.25 µg/ml	101.5%
2.50 µg/ml	91.3%
5.00 µg/ml	98.0%
10.0 µg/ml	82.1%
20.0 µg/ml	55.9%

Conclusions:

Interpretation of results: positive

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 05 to 17, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Physical state: clear colourless liquid
- Purity: 50.2%
- Lot/batch No.: #V6-612
- Storage: The test substance was obtained from the sponsor and was stored over the study period at room temperature as received (in an amber glass bottle). No physical changes indicative of instability were observed.

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: uvrB deletion mut., rfa mut., plasmid pKM101 (TA98, TA100), detection of base pair substitution mut. of hisG46 allele (TA1535, TA100),detection of frameshift mut. of hisD3052 allele (TA98), detection of frameshift mut. of hisC3076 allele (TA1537).

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: uvrB deletion mutation, rfa mutation, detection of frameshift mutations of hisD3052 allele,

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: rfa mut., hisG428 ochre mut. on the multicopy plasmid pAQ1, plasmid pKM101 enhancing the error-prone DNA repair system, detection of a wide variety of genetic damage affecting the hisG428 allele.

Species / strain / cell type:

S. typhimurium, other: TA 104

Additional strain / cell type characteristics:

other: uvrB deletion mut., rfa mut., plasmid pKM101 enhancing the error-prone DNA repair system, detection of a wide variety of genetic damage affecting the hisG428 allele.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix was obtained by mixing a series of cofactors (MgCl2, KCl, NADP, glucose-6-phosphate, Na2HPO4) with the S9 liver fraction of Aroclor 1254 treated male Sprague-Dawley rats.

Test concentrations with justification for top dose:

Preliminary toxicity pre-screen test (without S9 mix):50, 167, 500, 1670 and 5000 µg/plate
Ames test (with and without S9 mix):0.5, 1.67, 5.0, 16.7, 50 and 100 µg/plate

Vehicle / solvent:

All required dilutions were made with sterile de-ionized water (di-H20), Lot #122893, supplied by Pharmakon Research.
Dilutions were prepared the day of the test and used within two hours of preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterile de-ionized water, with and without S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: Methylglyoxal with strain T104: 250 µg/plate; 2-anthramine for strains TA 1535, TA 1537, TA 1538, TA 98, TA 100: 2.50 µg/plate

Details on test system and experimental conditions:

The study consisted of a toxicity pre-screen test (without S9 mix) followed by the Ames test (with and without S9 mix).

Toxicity pre-screen test:
Following incubation, the toxicity pre-screen test was conducted and the background lawn and spontaneous revertants were scored for normal, inhibited (i.e. no confluent bacterial lawn and/or presence of pindot colonies) and no growth.

Mutation test:
Mutation assays (main test and confirmatory test) were performed in triplicate cultures in all seven tester strains for each selected dose level of the test material, as well as for the positive and solvent controls.

For each test, 0.1 mL of the tester strain was added to 0.1 mL of test solution in 2 mL molten agar (supplemented with 0.5 mM histidine and 0.5 mM biotin) in test tubes. In case of metabolic activation, 0.5 mL of the S9 mix was added. The tubes were vortexed and the contents were poured onto minimal glucose plates and allowed to solidify. Within one hour the plates were inverted and incubated in the dark at 37°C over 48 h.

Evaluation criteria:

A positive result is defined as a statistically significant, dose-dependent increase in the nunber of histidine-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value. If the test material does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal.
A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine-independent revertants.

Statistics:

The revertant colonies were assessed by means of an Artek electronic colony counter interfaced with an IBM PC/AT computer. 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. In fact, solvent and positive controls were scored first, and test material treated cultures were scored only if the average negative control values were within historical ranges (mean X ± 2SD).
Statistical analyses were performed using the program developed by Snee and Irr (Mutation Res, 85:77-93, 1981), with significance established at the 95% confidence limit. Statistical analyses are performed only when a 50% increase in revertant frequency, relative to the concurrent negative controls, is observed. This 50% "trigger" was selected based upon the normal, spontaneous variation observed among replicate negative control cultures as well as spontaneous fluctuation observed in this laboratory among groups of cultures treated with a variety of test materials judged to be negative in this assay.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

SCREENING PRE-TEST:
A toxicity pre-screen test was conducted, without S9 mix, using TA 1538, TA 100 and TA 102 as tester strains. Following concentrations of the test substance were used: 50, 167, 500, 1670 and 5000 µg/plate. The results showed that the test substance affected the bacterial growth of all tester strains at all tested concentrations, resulting in inhibition to complete absence of growth.

CYTOTOXICITY IN THE MUTATION ASSAY:
In the mutation assay as such glutaraldehyde 50% was tested in all strains in presence and absence of S9 mix at doses of of 0.5, 1.67, 5.00, 16.7, 50 and 100 µg/plate. Inhibited growth again was observed in all tester strains at the doses 16.7, 50 and/or 100 µg/plate, with and without S9 mix.
In the confirmatory test, inhibited growth was observed in all tester strains at 50 and/or 100 µg/plate, with and without S9 mix.

REVERTANTS FREQUENCY IN THE MUTATION ASSAYS (MAIN AND CONFIRMATORY TEST):
A statistically significant and dose-dependent increase in the frequency of revertants (ca. 1.2 to 3.3 fold control values) was observed for the tester strains TA 1535, TA 98, TA 100, TA 102 and TA 104 in presence of S9 mix, and for TA 98, TA 100, TA 102 and TA 104 in absence of S9 mix. Such an increase (ca. 2.3 fold control values) also was reported for TA 1537 in the presence of S9 mix, but it was neither statistically significant nor dose-dependent. In the confirmatory test, a statistically significant and dose-dependent increase in the frequency of revertants (ca. 1.7 to 2.8 fold control values) was observed for the tester strains TA 98, TA 100, TA 102 and TA 104 in presence of S9 mix, and for TA 100, TA 102 and TA 104 in absence of S9 mix.

CONTROLS:
All positive and negative controls were within acceptable limits.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results: positive

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study report shows unit-errors in some tables. In fact, in these tables (e.g. page 23 of 166), µg/ml was inscribed by inadvertency instead of the correct ng/ml-unit. However, these errors did not affect the validity of this guideline-study, which was conducted in accordance with GLP.

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

- Physical state: clear colourless liquid
- Purity: 50.2%
- Composition of test material, percentage of components: Glutaraldehyde 50% aq. solution
- Lot/batch No.: #V6-612
- Storage: The test substance was obtained from the sponsor and was stored over the study period at room temperature as received (in an amber glass bottle). No physical changes indicative of instability were observed.

Target gene:

Xanthine-guanine phosphoribosyl transferase (XPRT) locus.
In fact, the wild-type CHO cells can metabolize 6-thioguanine to its toxic derivative as they possess XPRT activity. In case of XPRTmutation, the mutants are identifiable by giving them TG: presumptive mutants, due to the loss of XPRT activity are unable to convert the purine-analogue TG to its toxic monophosphate metabolite and therefore are resistant to the lethal effect of this metabolite.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cultured AS52 Chinese hamster ovary (CHO) cells were used.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared by mixing the S9 liver fraction from Aroclor 1254-treated male Sprague-Dawley rats with appropriate cofactors (MgCl2, KCl, NADP, glucose-6-phosphate, Na2HPO4).

Test concentrations with justification for top dose:

Preliminary toxicity pre-screen test (with and without S9 mix):
0.167, 0.5, 1.67, 5, 16.7, 50, 167, 500, 1670 and 5000 µg/mL
Main mutation assay, without S9-mix:
5, 16.7, 50, 100, 167, 250, 333, 500, 667, 833 and 1000 ng/mL
Main mutation assay, with S9-mix:
167, 500, 1670, 2500, 3330, 5000, 6670, 8330, 10,000, 13,300 and 16,700 ng/mL
Confirmatory test, without S9-mix:
50, 167, 333, 500, 1000, 1670,2500, 3330, 5000, 6670, 8330, 10,000, 13,300 and 16,700 ng/mL
Confirmatory test, with S9-mix:
167, 500, 1670, 2500, 3330, 5000, 6670, 8330, 10,000, 13,300 and 16,700 ng/mL
All test series were accompanied by untreated negative and solvent controls and by positive controls.

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(diH2O, 20 µL/mL)

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

First, a toxicity screening pre-test was conducted. The cells were examined for the reduction in colony forming ability, as parameter indicative of the cytotoxic potential of Sepacid GA 50 at defined test concentrations. The test was performed with and without S9-mix. The test concentrations for the main mutation test were selected on the basis of the results of the pre-screen test.

The way of application was similar for both, the toxicity pre-screen test and the main mutation assay.
The tester cells were plated in the appropriate medium at a density of about 8 x 105 cells/flask in 25 cm2 tissue culture flasks. They were incubated at 37 °C for 16 to 24 hours prior treatment. At the end of the incubation period and following a series of steps including medium exchanges and washing of the cultures, the cells, which were in exponential growth state (1 to 1.2 x 106 cells/flask), were treated with the test substance by addition of 50 to 100 µl of test or control solution (solvent: di-H2O) to each flask. The contents of the flasks were thoroughly mixed and incubated over 5 hours under standard conditions. At the end of the incubation period, the test or control solutions were replaced by medium via a series of washing and medium exchange steps. The cultures were subjected to a further incubation over 19 hours. In case of metabolic activation, 1 ml of S9 mix was added to the appropriate flasks. At the end of the 19-hour incubation period, the medium was aspired and the cultures were washed twice prior to being harvested by trypsinisation. 0.2 ml diluted cell aliquots were prepared and added to each of three 60-mm plates containing 5 ml of medium, resulting in a density of 200 cells/plate. The plates were incubated for 7 days and the cell colonies were fixed (ethyl alcohol), stained (crystal violet) and counted.
For the main assay, duplicate cultures were used for each test concentration as well as each positive of negative control.
Within the main mutation test and the confirmatory test, the following parameters were considered: the relative survival, the number of mutants, the cloning efficacity and the mutant frequency.

Evaluation criteria:

Toxicity pre-screen test:
The cells were examined for their ability to form colonies. Therefore, the reduction in colony forming ability of the cells was used as indicator for the cytotoxic potential of Sepacid GA 50 at defined test concentrations.

Main mutation assay:
Following treatment, the relative survival was determined for each culture. After a growth period of 8 days to allow expression of the mutant phenotype, 10E+6 from each culture were plated in medium containing 6-thioguanine to select mutant cells. The mutant frequency was expressed as TGr mutants/10E+6 clonable cells and was obtained by dividing the total number of mutant clones by the number of cells plated, corrected for the cloning efficiency (average of 3 plates) of the cells at the time of mutant selection.

Statistics:

The statistical assessment of the findings was based on Snee RD and Irr JD (Mut. Res. 85: 77-93, 1981).

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

for details see below

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

CONTROLS
All negative and positive controls were within acceptable ranges.

TOXICITY IN THE PRE-TEST
epacid GA 50 was found to be cytotoxic to AS52 Chinese hamster ovary cells in the absence and presence of S9 mix. In fact, without S9 mix, no more viable cells were present in the test cultures from 16.7 µg/ml test substance upwards (relative survival, 0%); in the presence of S9 mix, the same result was reported for test concentrations >=50 µg/ml (relative survival, 0.03%). For the negative untreated and solvent controls, a relative survival of 74 – 116% and 94 – 116% respectively was reported.

MAIN MUTATION TEST
The average mutant frequencies of the Sepacid treated cultures (excepted for 16,700 ng/ml with S9 mix, which was excluded from the mutation evaluation because of the high immediate cytotoxicity that was observed) ranged from 49.85 to 155.26 TGr mutants/1E6 clonable cells, versus 37 to 59 for the negative control cultures. The increase in mutant frequencies related to the treatment with Sepacid was statistically significant and dose-dependent in both cases, with and without S9 mix. The treatment-related increase was >= 2 fold control values, and represented net increases in average mutant frequencies of >= 30 TGr mutants/1E6 clonable cells.

CONFIRMATORY TEST
The re-evaluation of the test substance in a confirmatory test resulted in findings, which were in accordance with those reported above.

Remarks on result:

other: strain/cell type: AS52 cells

MAIN TEST WITHOUT S9 MIX

Control cultures, main results:

Compound	Dose (µg/ml)	Relative survival (%)	Cloning efficacity (%)	Average mutant frequency (mutants/10^6clonable cells)
Untreated	0	67.90	82.50	40.60
	0	93.14	76.17
Di-H2O (µl/ml)	20	125.39	79.50	37.01
	20	89.09	75.17
EMS	200	57.47	59.17	213.54**
	200	75.82	54.67

EMS, ethyl methanesulfonate, positive control; *, p<0.05; **, p<0.01

Treated cultures, main results:

Sepacid50 Dose (ng/ml)	Relative survival (%)	Cloning efficacity (%)	Average mutant frequency (mutants/10^6clonable cells)
5	100.81	71.00	91.71**
5	101.30	64.00
16.7	80.96	67.67	77.10*
16.7	68.92	67.17
50	104.43	64.83	71.13*
50	66.41	67.17
100	65.11	69.17	75.85*
100	110.68	71.83
167	102.41	72.83	87.64**
167	51.48	69.00
250	92.27	68.67	49.85
250	120.47	69.83
333	74.25	70.17	68.74
333	99.94	64.00
500	102.63	73.00	108.55**
500	79.99	65.67
667	89.97	66.00	102.12**
667	87.63	71.67
833	76.07	93.17	70.98*
833	76.01	92.83
1000	76.50	88.33	68.71
1000	77.00	85.00

 

MAIN TEST WITH S9 MIX

Control cultures, main results:

Compound	Dose (µg/ml)	Relative survival (%)	Cloning efficacity (%)	Average mutant frequency (mutants/10^6clonable cells)
Untreated	0	116.51	67.83	53.40
	0	103.84	59.50
Di-H2O (µl/ml)	20	92.28	69.17	59.05
	20	111.85	85.00
DMN	100	35.04	51.83	304.47**
	100	40.20	55.17

DMN, Dimethylnitrosamine, positive control; *, p<0.05; **, p<0.01

Treated cultures, main results:

Sepacid50 Dose (ng/ml)	Relative survival (%)	Cloning efficacity (%)	Average mutant frequency (mutants/10^6clonable cells)
167	99.72	66.00	97.52**
167	111.05	52.00
500	111.14	62.83	72.93
500	135.59	63.33
1670	85.14	50.17	79.37*
1670	110.53	66.17
2500	95.30	68.00	81.13*
2500	111.98	72.00
3330	111.37	56.00	116.80**
3330	93.65	44.00
5000	92.39	57.17	134.21**
5000	91.59	53.83
6670	43.78	63.00	81.61*
6670	49.55	54.00
8330	65.16	66.33	155.26**
8330	52.68	55.50
10,000	51.24	70.83	133.29**
10,000	18.38	58.83
13,300	21.58	56.50	120.47**
13,300	15.66	58.83
16,700	8.33	56.67	96.80 (excluded from mutation evaluation because of high tox.)
16,700	3.33	57.17

CONFIRMATORY TEST WITHOUT S9 MIX

Control cultures, main results:

Compound	Dose (µg/ml)	Relative survival (%)	Cloning efficacity (%)	Average mutant frequency (mutants/10^6clonable cells)
Untreated	0	88.91	61.67	32.67
	0	86.74	63.83
Di-H2O (µl/ml)	20	90.73	72.67	32.70
	20	87.23	66.67
EMS	200	59.60	56.33	199.38**
	200	66.10	47.67

EMS, ethyl methanesulfonate, positive control; *, p<0.05; **, p<0.01

 

Treated cultures, main results:

Sepacid50 Dose (ng/ml)	Relative survival (%)	Cloning efficacity (%)	Average mutant frequency (mutants/10^6clonable cells)
50	73.87	58.00	22.46
50	60.37	78.67
167	66.88	73.83	46.10*
167	79.56	56.33
333	54.70	71.50	30.38
333	67.59	59.50
500	81.15	66.17	50.38*
500	55.90	64.67
1000	62.11	64.50	37.99
1000	64.62	81.83
1670	18.01	78.67	48.68*
1670	17.78	84.00
2500	2.75	66.83	88.83 (excluded from mutation evaluation because of high tox.)
2500	1.47	65.83
3330	0.26	68.67	213.86 (excluded from mutation evaluation because of high tox.)
3330	0.15	67.17

Remark: due to the extreme immediate cytotoxicity, the test concentrations > 3330 ng/ml were not taken into consideration

CONFIRMATORY TEST WITH S9 MIX

Control cultures, main results:

Compound	Dose (µg/ml)	Relative survival (%)	Cloning efficacity (%)	Average mutant frequency (mutants/10^6clonable cells)
Untreated	0	108.18	91.50	20.86
	0	119.36	75.00
Di-H2O (µl/ml)	20	108.15	75.50	21.82
	20	110.69	75.67
DMN	100	36.13	52.50	312.26**
	100	37.10	39.33

DMN, Dimethylnitrosamine, positive control; *, p<0.05; **, p<0.01

Treated cultures, main results:

Sepacid50 Dose (ng/ml)	Relative survival (%)	Cloning efficacity (%)	Average mutant frequency (mutants/10^6clonable cells)
167	91.13	61.33	31.29
167	74.82	56.17
500	101.75	54.67	29.15
500	83.01	67.00
1670	72.23	71.00	25.94
1670	66.58	68.00
2500	84.13	66.83	37.06
2500	85.82	68.33
3330	81.40	69.83	23.75
3330	88.56	69.17
5000	77.34	56.33	40.94
5000	75.21	63.33
6670	53.95	69.67	56.22**
6670	42.24	55.33
8330	43.13	53.33	68.85**
8330	44.93	60.00
10,000	19.03	65.67	38.87
10,000	19.71	57.67
13,300	10.80	44.17	63.18**
13,300	10.13	44.50
16,700	2.72	50.33	75.48 (excluded from mutation evaluation because of high tox.)
16,700	0.47	61.00

Conclusions:

Interpretation of results: positive

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Glutaraldehyde was not clastogenic in an in vivo micronucleus assay conducted according to guideline and GLP.

Glutaraldehyde was non-mutagenic to germ cells in the Drosophila SLRL test

Glutaraldehyde was not genotoxic in an unscheduled DNA synthesis (UDS) test with mammalian liver cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

- Physical state: homogeneous colourless-clear liquid
- Analytical purity: 50.5%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: ca. 50% glutaraldehyde in water
- Lot/batch No.: 50-4402
- Storage condition of test material: refrigerator (under N2 conditions)

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: about 10 to 12 weeks
- Weight at study initiation: about 256 g
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon cages, type III
- Diet (e.g. ad libitum): standardized pelleted feed (Kliba-Haltungsdiät/Provimi Kliba SA, Kaiseraugst, Switzerland) , ad libitum
- Water (e.g. ad libitum): drinking water from bottles, ad libitum
- Acclimation period: not specified
- Other: feed, water and bedding were analyzed for contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs

Route of administration:

oral: gavage

Vehicle:

purified water

Details on exposure:

PREPARATION OF THE TEST SOLUTIONS
All test solutions were prepared once weekly before administration. The amount of test material or volume to be administered was related to the specific weight of the individual animals on the day of treatment.

PRE TEST FOR DETERMINATION OF THE MTD
In a pretest for the determination of the acute oral toxicity (male and female rats), deaths were observed down to a dose of 400 mg/kg body weight (1 out of 14 animals were found dead the day after test substance administration). The clinical signs observed were piloerection, squatting posture, gasping respiration and bloody snout and the general state of the animals was poor. However, there were no distinct symptomatic differences between the male and female animals. Thus, only male animals were used for the main experiment.
Therefore, a dose of 400 mg/kg body weight was selected as the maximal tolerated dose level (MTD) for the main assay, and 200 mg/kg body weight were administered as further dose; both dosages refer to the aqueous test substance and not to the active ingredient, i.e.
Glutaraldehyde (about 50%).

UDS TEST
The test series consisted of following groups:
Control group 1 and 2: vehicle controls with animals receiving 10 mL water/kg bw.
Test groups 3 and 4: animals receiving 200 mg test substance/kg bw, application volume 10 mL/kg bw of a 2% test solution.
Test groups 5 and 6: animals receiving 400 mg test substance/kg bw, application volume 10 mL/kg bw of a 4% test solution.
All the animals were treated once and were examined for clinical signs of toxicity and body weight change.
The animals were sacrificed and their livers perfused 3 hours and 14 hours after treatment.

Duration of treatment / exposure:

Not applicable as single application by gavage

Frequency of treatment:

Single application

Post exposure period:

3 and 14 hrs after single dose administration

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

the test concentrations refer the test substance as such and not to the active ingredient

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

the test concentrations refer the test substance as such and not to the active ingredient

No. of animals per sex per dose:

3 animals were used per group.

Control animals:

yes

Positive control(s):

As a positive control, 50 mg of 2-acetylaminofluorene/kg bw, suspended in corn oil, was administered to the animals once orally in a volume of 10 mL/kg body weight.
The stability of 2-AAF is well-defined under the selected culture conditions, since 2-AAF is a well-established UDS-inducing agent.

Tissues and cell types examined:

The hepatocytes were isolated from the liver of the treated animals 3 and 14 hours after treatment according to the procedure described by Butterworth BE et al., Mutat. Res. 189(2): 123-133, 1987). The main steps were as follows:
- Anesthetization of the animals with Metofane by inhalation;
- Liver perfusion with ethylene glycoI-bis(ß-amino ethyl ether) N,N,N',N',-tetraacetic acid (EGTA) solution followed by collagenase solution;
- Obtention of a cell suspension;
- Filtration, centrifugation steps with removal of supernatant and obtention of cell pellets;
- Resuspension of the cell pellets to yield single ceIl suspensions.

The hepatocytes were examined for cell viability as measured by the trypan blue exclusion technique, and for conspicuous changes in cell morphology.
DNA damage and repair was measured by incorporation of 3H-Thymidine using autoradiography technique. For this purpose about 400,000 viable cellson coverslips were seeded per well and 6 wells per animal were used for the UDS assay. For labeling, the initial medium was replaced by labeling medium, and the cells were incubated for 4 hours. The cells on the coverslips were then fixed, rinsed and air-dried before being mounted cell side up an glass slides using Corbit-Balsam.
Autoradiography was carried out according to the procedure of Butterworth et al. (see above), and for this purpose the slides were coated with KODAK NTB-2 photographic emulsion.
about 5 -10 seconds.
In fact, for microscopical evaluation and quantification of UDS, a total of 100 cells/animal was examined.
Following parameters were considered:
- the net nuclear grain (NNG) count/cell,
- the mean nuclear grain (NG) count,
- the mean cytoplasmic grain count (CG) count,
- the mean net nuclear grain count,
- the % of cells in repair (cells with NNG >= 0; cells with NNG >= 5).

Evaluation criteria:

Acceptance criteria:
-Clearly negative results in the untreated and in the vehicle controls in the range of historical control data.
-Clearly positive results in the positive control group (>=30% of cells in repair, as mean of 3 animals) in the range of historical control data.
-Viability (trypan blue staining) of at least 70% in hepatocytes from negative control animals.
Evaluation criteria:
-Positive response: 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.
-Marginal response: a response is considered marginal when the dose-related increase in percentage of cells in repair is >= 5 outside the values of both the concurrent negative control and the historical control (>= 5 < 20), and when the dose-related increase in mean number of NNG counts is near to but without exceeding 0. In this case, an additional confirmatory test is needed.
-Negative response: a negative response implicates that both, the NNG counts and the percentage of cells in repair are within the range of negative control.

Statistics:

Due to clear negative findings, a statistical evaluation was not carried out.

Sex:

male

Genotoxicity:

negative

Remarks:

The microscopical evaluation and quantification of UDS showed that the results obtained for the GA-treated animals were within the range of negative controls, as can be seen from the table included below.

Toxicity:

yes

Remarks:

Evident signs of toxicity were seen in animals treated with the test material at both test doses and included piloerection, squatting posture and bloody snout.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

For all treated animals including those serving for positive control, the cell viability after 3 and 14 hours was within 94 - 102% of control.
The microscopical evaluation and quantification of UDS showed that for the negative controls, the frequencies of mean nuclear grain counts were within historical control range.
For the positive controls, the increase in unscheduled DNA synthesis was as expected.
The results obtained for the glutaraldehyde-treated animals were within the range of negative controls, as can be seen from following table.

Test group	Negative Control.	Protectol GDA 200 mg/kg bw	Protectol GDA 400 mg/kg bw	Positive control
Cells harvested after 3 hours following treatment
NG counts*	6.79 +/- 0.99	7.79 +/- 1.26	6.47 +/- 0.27	21.23 +/- 1.82
CG counts*	12.07 +/- 1.46	14.61 +/- 0.26	12.45 +/- 0.19	15.46 +/- 1.40
NNG counts**	- 5.28 +/- 0.65	- 6.82 +/- 1.02	-5.98 +/- 0.42	5.77 +/- 2.57
% cells in repair (NNG >= 0)	8.33 +/- 0.58	6.00 +/- 3.61	3.33 +/- 1.53	80.33 +/- 9.07
% cells in repair (NNG >= 5)	0.00 +/- 0.00	0.00 +/- 0.00	0.00 +/- 0.00	50.67 +/- 14.98
Cells harvested after 14 hours following treatment
NG counts*	7.88 +/- 2.31	7.66 +/- 0.43	6.48 +/- 0.64	19.82 +/- 2.38
CG counts*	13.01 +/- 2.70	13.67 +/- 1.17	11.57 +/- 1.36	13.63 +/- 1.62
NNG counts**	-5.13 +/- 0.61	-6.01 +/- 0.76	-5.09 +/- 0.72	6.20 +/- 1.04
% cells in repair (NNG >= 0)	8.33 +/- 0.58	6.33 +/- 4.51	6.33 +/- 2.08	80.00 +/- 3.61
% cells in repair (NNG >= 5)	0.00 +/- 0.00	0.00 +/- 0.00	0.00 +/- 0.00	52.33 +/- 4.73

NG, nuclear grains; CG, cytoplasmic grains, NNG, net nuclear grains; *, mean per group (3 animals) with standard deviation

Reference 2

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientifically acceptable data.

Principles of method if other than guideline:

Mutations in the X-chromosome of D. melanogaster are phenotypically expressed in males carrying the mutant gene. When the mutation is lethal in the hemizygous condition, its presence is inferred from the absence of one class of male offspring out of the two that are normally produced by a heterozygous female. The SLRL test takes advantage of these facts by means of specially marked and arranged chromosomes.
Three tests were conducted according to the methods of Yoon JS et al. (Environ Mutagen. 7(3):349-367, 1985) and of Zimmering S et al. (Environ. Mol. Mutagen. 14(4):245-251, 1989.

GLP compliance:

not specified

Type of assay:

Drosophila SLRL assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Glutaraldehyde 50% aq. solution, from Union Carbide (South Charleston, WV),
- Physical state: clear colourless liquid
- Analytical purity: 50.0%
- Lot/batch No.: 95296
- Stability under test conditions: stability verified for 2 weeks when stored in the dark at temperatures up to 25 °C (stability tested by the Midwest Research Institute, Kansas City, MO).
- Storage condition of test material: in the dark at temperatures up to 25 °C

Species:

Drosophila melanogaster

Strain:

other: Canton-S wild-type

Sex:

male

Details on test animals or test system and environmental conditions:

Age of the flies at test initiations:
First approach: Adult male flies (24 to 72 h old)
Second approach: Eggs
Third approach: Adult male flies (<= 24 h old)

Route of administration:

other: single injection or oral, depending on the testing approach (see details on exposure)

Vehicle:

water, ethanol or sucrose, depending on the testing approach (see details on exposure)

Details on exposure:

Three testing approaches were considered:
First approach, according to Yoon JS et al. (Environ Mutagen. 7(3):349-367, 1985):
Adult male flies (24 to 72 h old) received single injection of the test substance and were allowed to recover for 24 hours. Each of the treated males was then mated with 3 Basc females for 3 days; the fresh females were given for mating at 2-days intervals to produce 3 matings of 3, 2 and 2 days. The offspring of the females were scored for lethal effects corresponding to the effects on mature sperm, mid or late stage spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment. The heterozygous female offspring were mated individually to their brothers and were then placed in individual vials. The F1 females of the same parental male were kept together to identify clusters. In the next generation the progeny from each separate dose was scored for phenotypically wild-type males. The presence of less than 5% of the expected number of wild-type males per vial after 17 days was indicative of a sex-linked recessive lethal mutation that had occurred in a germ cell of the parental male following treatment with glutaraldehyde.

Second approach according to Zimmering S et al. (Environ. Mol. Mutagen. 14(4):245-251, 1989):
Adult male and female flies were mated and the eggs were placed into vials containing corn meal food with glutaraldehyde in solvent (5% ethanol). The adult emergent males (ca. 24 h old) were mated with 2 successive harems of 3 to 5 Basc females to produce 2 single-day broods. The offspring of these females were scored for lethal effects corresponding to the effects on mature sperm, mid or late stage spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment. The heterozygous female offspring were mated individually to their brothers and were then placed in individual vials. The F1 females of the same parental male were kept together to identify clusters. In the next generation the progeny from each separate dose was scored for phenotypically wild-type males. The presence of less than 5% of the expected number of wild-type males per vial after 17 days was indicative of a sex-linked recessive lethal mutation that had occurred in a germ cell of the parental male following treatment with glutaraldehyde.

Third approach according to Yoon JS et al. (Environ Mutagen. 7(3):349-367, 1985):
Adult male flies (<= 24 h old) were fed a solution of test substance in 5% sucrose for 3 days. A control group consisted of flies fed with 5% sucrose only. Each of the treated males was then mated with 3 Basc females for 3 days; the fresh females were given for mating at 2-days intervals to produce 3 matings of 3, 2 and 2 days. The offspring of these females were scored for lethal effects corresponding to the effects on mature sperm, mid or late stage spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment. The heterozygous female offspring were mated individually to their brothers and were then placed in individual vials. The F1 females of the same parental male were kept together to identify clusters. In the next generation the progeny from each separate dose was scored for phenotypically wild-type males. The presence of less than 5% of the expected number of wild-type males per vial after 17 days was indicative of a sex-linked recessive lethal mutation that had occurred in a germ cell of the parental male following treatment with glutaraldehyde.

Duration of treatment / exposure:

First approach: Single injection
Second approach: Oral feed over the larval development; corn meal food with glutaraldehyde in solvent (5% ethanol).
Third approach: Oral feed over 3 days; solution of test substance in 5% sucrose.

Frequency of treatment:

See above

Post exposure period:

See above

Dose / conc.:

3 000 ppm

Remarks:

First approach

Dose / conc.:

4 000 ppm

Remarks:

First approach

Dose / conc.:

3 500 ppm

Remarks:

Second approach

Dose / conc.:

7 500 ppm

Remarks:

Third approach

Dose / conc.:

10 000 ppm

Remarks:

Third approach

No. of animals per sex per dose:

Not applicable

Control animals:

yes

Statistics:

The SLRL data were assessed statistically by simultaneous comparison with the concurrent and historical controls using a normal approximation to the binomial test according to Margolin BH et al. (Environ. Mutagen. 5: 705-716, 1983).

Sex:

male

Genotoxicity:

negative

Remarks:

For all 3 testing approaches, the percentage of sex-linked lethal mutations was within control range (0.06 – 0.24%).

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

SLRL TEST WITH DROSOPHILA, SUMMARY OF MAIN FINDINGS:

Drosophila SLRL test, first approach (single injection of GA in adult male flies)
Dose	Deaths	Sterility	Sex-linked lethal mutation	Result of the test
0 ppm	-	-	0.06 – 0.24%	Negative
3000 ppm	2%	0%	0.22%
4000 ppm	22%	54%	0.10%
Drosophila SLRL test, second approach (oral feed, larvae)
Dose	Deaths	Sterility	Sex-linked lethal mutation	Result of the test
0 ppm	-	-	0.08%	Negative
3500 ppm	10%	0%	0.11%
Drosophila SLRL test, third approach (oral feed, adult male flies)
Dose	Deaths	Sterility	Sex-linked lethal mutation	Result of the test
0 ppm	-	-	0.07 – 0.21%	Negative
7500 ppm	27%	37%	0.10%
10000 ppm	68%	2%	0.00%

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

- Physical state: clear colourless liquid
- Analytical purity: about 50% glutaraldehyde
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: 50 % glutaraldehyde in water
- Lot/batch No.: #V6-612
- Stability under test conditions: No physical changes indicative of instability were observed under the storage conditions reported below.
- Storage condition of test material: The test substance was obtained from the sponsor and was stored over the study period at room temperature as received (an amber glass bottle).

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA 01887
- Age at study initiation: the mice were about 7 weeks old
- Weight at study initiation: the mice weighed between 28 and 33 g for males and between 21 and 26 g for females
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in stainless steel wire mesh cages, at maximum five per cage according to sex and dose group.
- Diet (e.g. ad libitum): throughout the study, mice were fed Harlan Teklad Rodent Diet
- Water (e.g. ad libitum): fresh tap water was available ad libitum
- Acclimation period: the mice were acclimated to laboratory conditions for 28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 4
- Humidity (%): 55% ± 15
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h

Route of administration:

intraperitoneal

Vehicle:

Deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Aliquots of Sepacid GA 50 were weighed in separate tared mortars. The appropriate amount of deionized water was added and mixed thoroughly.
The test solutions were prepared fresh prior to dosing and were stirred continuously during dosing.

TREATMENT:
The whole study consisted of a dose range-finding study (DRF; for determination of the adequate test doses for the MNT), which was followed by the micronucleus test (MNT); an additional confirmatory test using female animals only was added. For treatment the mice received single application of the test solution via i.p. injection; the application volume was 10 mL/kg bw.

EXAMINATION:
The animals were observed for clinical symptoms of toxicity, and after necropsy, the femoral bone marrow tissue was removed and prepared for erythrocytes examination.

CYTOGENIC ANALYSIS:
For cytogenic analysis, 1000 PCE were scored for MPCE and following parameters ratios were determined:
- Micronucleated polychromatic erythrocytes/polychromatic erythrocytes (MPCEs/PCEs)
- Polychromatic erythrocytes/normochromatic erythrocytes ratio (PCEs/NCEs) for 1000 erythrocytes

Duration of treatment / exposure:

Not applicable as the animals received single i.p. injections as treatment.

Frequency of treatment:

Single i.p. injection

Post exposure period:

24, 48 and 72 h after treatment

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Remarks:

in drinking water, Dose Range Finder (DRF)

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

in drinking water, Dose Range Finder (DRF)

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

in drinking water, Dose Range Finder (DRF)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

in drinking water, Dose Range Finder (DRF)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

in drinking water, Dose Range Finder (DRF)

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

in drinking water, Dose Range Finder (DRF)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

in drinking water, Dose Range Finder (DRF)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

in drinking water, Dose Range Finder (DRF)

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Remarks:

in drinking water, Dose Range Finder (DRF)

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

in drinking water, Micronucleus Test (MNT)

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

in drinking water, Micronucleus Test (MNT)

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

in drinking water, Micronucleus Test (MNT)

No. of animals per sex per dose:

DRF: 2/sex/group, 9 treated groups and one negative control group (deionized water)
MNT: 5/sex/group, 9 treated groups (i.e. 3 groups/dose level), 3 negative control groups (deionized water), one positive control group (triethylenemelamine, TEM, 0.5 mg/kg bw)

Control animals:

yes

Positive control(s):

Triethylenemelamine, TEM, 0.5 mg/kg bw, was used as positive control substance for the MNT.

Tissues and cell types examined:

The femoral bone marrow were examined for polychromatic erythrocytes (PCEs), micronucleated polychromatic erythrocytes (MPCEs) and for normochromatic erythrocytes /NCEs).

Details of tissue and slide preparation:

The preparation of the bone marrow was based on the method of Schmid W (Mut. Res. 19:109-117, 1975).
The main steps of the tissue preparation were as follows:
- Dissection/removal of the soft tissues from the femora
- Extraction of the bone marrow and sampling in 5 ml round bottom culture tubes containing fetal bovine serum
- Mixing and centrifugation of the bone marrow suspension
- Removal of the supernatant and resuspension of the precipitate
- Application of a drop of the suspension onto slides for microscopy
- Rapid drying of the slides on a slide warmer (ca. 56°C)
- Short dipping of the slides in absolute methanol and air-drying
- Staining of the preparations on the slides with a modified Wrights Stain Pak (4481; polychrome methylene blue-eosin)
- Air-drying
- Coverslipping of the slides.

Evaluation criteria:

The results of the assay are acceptable if:
- In the vehicle control, the number of MPCEs/1000 PCES/mouse is < 6 and the spontaneous rate of MPCEs is <0.25% per dose group;
- The positive control produces a statistically significant increase (p =<0.05 as compared to the frequency of the negative control);
- At least seven mice per group survive the treatment.

Statistics:

One-tailed t-tests were used to make pairwise comparisons between each treatment group and its concurrent negative control for statistically significant increases in the number of MPCEs. The proportion of PCEs per 1000 erythrocytes per mouse was evaluated by pairwise two-tailed t-tests after an arcsin transformation was performed. Statistical significance was judged at p=<0.05 and p=<0.01 levels.
All comparisons were made for each sacrifice time separately, comparing the treated group versus the negative control group.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Additional information on results:

DRF (0.5 to 2500 mg/kg bw):
All mice treated with doses >= 100 mg/kg bw died within 24 hours; at 50 mg/kg bw, 1 male and 1 female died after 72 h. No mortality was observed in lower dosed groups. Symptoms of toxicity were observed at 5, 25 and 50 mg/kg bw and mainly included writhing, abnormal gait, distended abdomen and piloerection. At the lowest tested dose of 0.5 mg/kg bw, no such symptoms were seen.
MNT (2, 10 and 20 mg/kg bw):
As expected, no mortality was observed at the chosen test doses. In fact, symptoms of toxicity included piloerection (all doses), writhing (all doses) and abnormal gait (at 10 and 20 mg/kg bw). No such symptoms were seen in the controls (both, positive and negative).

Genotoxicity:
For details, see the tables below.
A statistically significant increase in the frequency of MPCE in mice treated with 10 mg/kg bw Sepacid GA 50 was observed at sacrifice time point 24 h, which was mainly due to the females. A statistically significant decrease of the PCE/NCE ratio was reported for the females at sacrifice time point 24 h; when combined together with the results for the males, a similarly significant decrease was observed at 20 mg/kg bw after 24 and 48 hours. The decrease of the PCE/NCE ratio was indicative of the toxicity of the test substance to bone marrow, even though it did not increase the frequency of MPCE.
Within the confirmatory test, the statistically significant findings of the main micronucleus test could neither be reproduced nor confirmed. Thus, it was concluded that since neither a reproducible statistically significant increase nor a clear dose-related increase in the frequency of MPCE were observed, Sepacid GA 50 was non-clastogenic in the in vivo micronucleus test.

MICRONUCLEUS TEST

Mean percentage of micronucleated polychromatic erythrocytes (MPCE) for male (M) and female (F) mice at time point 24 h
Sex	TS	Dose (mg/kg bw)			Total MPCE/ 5000 PCE^b		Range			% MPCE
M	DeH2O	0			1		0-1			0.02+/-0.045
	Sepacid	2			4		0-2			0.08+/-0.084
	Sepacid	10			3		0-2			0.06+/-0.089
	Sepacid	20			5		0-3			0.10+/-0.123
	TEM^a	0.5			93		8-29			1.86+/-0.783**
F	DeH2O	0			4		0-2			0.08+/-0.084
	Sepacid	2			1		0-1			0.02+/-0.045
	Sepacid	10			9		1-3			0.18+/-0.084*
	Sepacid	20			1		0-1			0.02+/-0.045
	TEM^a	0.5			95		17-21			1.90+/-0.187**
*, one-tailed t-tests, p<=0.05; **, p<=0.01;a, positive controltriethylenemelamine; b, 1000 PCE per mouse and 5000 per sex and group
Mean percentage of micronucleated polychromatic erythrocytes (MPCE) for male (M) and female (F) mice at time point 48 h
Sex	TS	Dose (mg/kg bw)		Total MPCE/ 5000 PCE^a		Range			% MPCE
M	DeH2O	0		4		0-2			0.08+/-0.084
	Sepacid	2		0		0-0			0.00+/-0.000
	Sepacid	10		1		0-1			0.02+/-0.045
	Sepacid	20		4		0-3			0.08+/-0.130
F	DeH2O	0		2		0-1			0.04+/-0.055
	Sepacid	2		1		0-1			0.02+/-0.045
	Sepacid	10		2		0-1			0.04+/-0.055
	Sepacid	20		6		0-2			0.12+/-0.084
*, one-tailed t-tests , p<=0.05; **, p<=0.01; a,1000 PCE per mouse and 5000 per sex and group
Mean percentage of micronucleated polychromatic erythrocytes (MPCE) for male (M) and female (F) mice at time point 72 h
Sex	TS	Dose (mg/kg bw)	Total MPCE/ 5000 PCE^a					Range			% MPCE
M	DeH2O	0	2					0-1			0.04+/-0.055
	Sepacid	2	4					0-1			0.08+/-0.045
	Sepacid	10	2					0-2			0.04+/-0.089
	Sepacid	20	3					0-2			0.06+/-0.089
F	DeH2O	0	4					0-3			0.08+/-0.130
	Sepacid	2	3					0-1			0.06+/-0.055
	Sepacid	10	2					0-2			0.04+/-0.089
	Sepacid	20	2					0-1			0.04+/-0.055
*, one-tailed t-tests , p<=0.05; **, p<=0.01; a,1000 PCE per mouse and 5000 per sex and group

PCE/NCE ratios for male (M) and female (F) mice at time point 24 h
Sex	TS	Dose (mg/kg bw)	Range	Mean +/- SD
M	DeH2O	0	1.304-2.040	1.681+/-0.306
	Sepacid	2	0.988-1.833	1.373+/-0.340
	Sepacid	10	1.294-1.597	1.494+/-0.125
	Sepacid	20	0.901-1.994	1.397+/-0.400
	TEM	0.5	0.842-1.907	1.340+/-0.414
F	DeH2O	0	1.008-2.571	1.619+/-0.596
	Sepacid	2	1.899-3.504	2.541+/-0.819
	Sepacid	10	0.748-1.915	1.352+/-0.452
	Sepacid	20	0.667-1.174	0.954+/-0.194*
	TEM	0.5	0.681-2.257	1.407+/-0.637
*, one-tailed t-tests , p<=0.05; **, p<=0.01
PCE/NCE ratios for male (M) and female (F) mice at time point 48 h
Sex	TS	Dose (mg/kg bw)	Range	Mean +/- SD
M	DeH2O	0	1.336-1.833	1.647+/-0.200
	Sepacid	2	0.848-2.257	1.552+/-0.526
	Sepacid	10	1.336-2.584	1.905+/-0.456
	Sepacid	20	0.250-1.793	1.114+/-0.570
F	DeH2O	0	1.053-1.841	1.551+/-0.327
	Sepacid	2	1.336-1.786	1.548+/-0.194
	Sepacid	10	1.049-2.333	1.651+/-0.486
	Sepacid	20	0.416-2.040	1.138+/-0.643
*, one-tailed t-tests , p<=0.05; **, p<=0.01

CONFIRMATORY TEST

 

Mean percentage of micronucleated polychromatic erythrocytes (MPCE) for female mice in the confirmatory test ( 24 h)
Sex	TS	Dose (mg/kg bw)	Total MPCE/ 5000 PCE^a	Range	% MPCE
F	DeH2O	0	6	0-2	0.06+/-0.07
	Sepacid	10	9	0-2	0.09+/-0.088
*, one-tailed t-tests , p<=0.05; **, p<=0.01; a,1000 PCE per mouse and 5000 per sex and group

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Ames

Several Ames tests have been performed. There is one GLP compliant guideline Ames study available. This study by Pharmakon (1994), performed in accordance with guideline EPA OPP 84 -2, tested glutaraldehyde (50%) up to cytotoxic concentrations (0 -100 µg/plate) in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100, TA 102, and TA 104 with and without the addition of S9 -mix. It was observed that glutaraldehyde was mutagenic to several S. typhimurium strains with and without metabolic activation. Positive results were also obtained by Vergnes (2002), NTP (1993), Marnett (1985), Jung (1992), Dillon (1998), Müller (1993), JETOC (2005) and Watanabe (1998). Two studies showed negative results: Slesinski (1983) and Hemminki et al (1980). The latter only tested E. coli WP2 uvr A without metabolic activation. Overall it can be concluded that glutaraldehyde is mutagenic to bacteria in an Ames test.

Chromosome aberration test and micronucleus assay

Four chromosome aberration tests were performed. The study by BASF (2002) was performed in accordance with OECD Guideline 473 and following GLP. In this study, Chinese hamster lung fibroblasts (V79) were exposed to glutaraldehyde (50%) at concentrations of 0.25, 0.5 and 1 µg/mL (without S9 mix) and 2.5, 5 and 10 µg/mL (with S9 mix). Exposure to glutaraldehyde caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases (with and without gaps), resulted in a weak suppression of the mitotic activity, and slightly affected cell growth in the concentration-range tested. No aneugenic activity on V79 cells was seen. Positive results were also observed, in CHO cells, by Galloway (1985). However, in a study by Vergnes (2002) and in a study by Hikiba (2005) no increase in chromosomal aberrations in CHO cells or SHE cells, respectively, either in the presence or absence of metabolic activation was observed. In a micronucleus assay performed by Speit (2008) using V79 cells a slight but concentration-related and statistically significant increase in the micronucleus frequency was measured in the absence of metabolic activation. Overall it can be concluded that glutaraldehyde is clastogenic in mammalian cells.

Mammalian Cell Gene Mutation Tests

Seven in vitro mammalian cell gene mutation tests are available. In five of these tests, Chinese hamster ovary (CHO) cells were used and mutations in the XPRT gene were determined in order to draw a conclusion on the mutagenicity of glutaraldehyde (50%). In the sixth study, human lymphoblastoid cells (TK6) were used and mutagenicity of glutaraldehyde (25%) was tested in the absence of a metabolic activation system (St. Clair et al., 1991). In the seventh study, mouse lymphoma L5178Y cells were tested in the absence of metabolic activation (McGregor 1988). Mixed results were obtained; four out of seven studies concluded that glutaraldehyde is not mutagenic in the presence and absence of S9 -mix (Vergnes (2002), BRRC (1981), Carnegie-Mellon (1980), and Slesinski (1993)). The study performed by Pharmakon (1994) concluded that glutaraldehyde is mutagenic in the presence and absence of a metabolic activation system. St Clair et al. (1991) and McGregor et al. (1988) demonstrated mutagenic activity in the absence of S9 mix.

Comet assay

A standard comet assay and a modified comet assay for the detection of DNA-protein crosslinks (DPX) was performed using Chinese hamster lung fibroblasts (V79) (Speit 2008). In the standard comet assay, cells were exposed to 0 (control), 0.5, 1, 2, 5, 10 and 20 µM. Determination of DPX by the reduction of induced DNA migration was accomplished by irradiating GA-treated cultures with 2 Gy gamma-rays and exposure levels of 0.5, 1, 2, 5, 10, 20, 40 and 50 µM glutaraldehyde. Under standard conditions no increase in DNA migration (i.e. no indication for a strand-breaking effect) but a trend to reduced DNA migration at higher concentrations (suggestive of DPX) was observed. After DNA migration induced by gamma radiation, the test substance reduced DNA migration in the comet assay in a concentration related manner (significant at 10 µM and above). The induction of DPX as measured by the comet assay occurred in parallel to the cytotoxic effect of GA measured as reduction of cell growth.

In a comet assay performed by Vock (1999), pulsed-field gel electrophoresis (PFGE) was utilized to assess the potential of the test substance in human lung epithelial cells (A549), to cause the formation of DNA double strand breaks (DSB). Dose-time-response curves (sampling time 8, 24, 72 h) for survival (MTT assay) and DSB formation were established, and the size distribution of the formed DNA fragments analysed. At 8 h and 24 h the DSB induced by glutaraldehyde were a mirror-like image of induction of cell death, while at 72 h at 100 µM, glutaraldehyde induced DSB without any impairment of cell viability. However, this late process of DNA fragmentation was considered a consequence of cell death since a clearly cytotoxic molecular size distribution below 0.5 Mbp was observed and it has been shown that lethally damaged cells can show normal metabolic activity for several days, allowing reduction of the MTT dye in cells predestined to cell death.

In another comet assay (Kuchenmeister, 1998) glutaraldehyde was studied in freshly isolated rat hepatocytes. The observed mean tail moment did not differ from the control data. However, in approximately 90% of the DNA spots, high density areas became visible at concentrations ≥ 5 mg/mL, while cell viability was not affected as measured by Trypan blue exclusion. The observed areas could be the result of the DNA crosslinking activity. Nonetheless, a conclusion on genotoxicity could not be drawn based on the results of this study.

Miscellaneous

Two well performed SCE tests are available. Chinese hamster Ovary (CHO) cells (NTP 1993) and Chinese hamster lung fibroblasts (V79) (Speit et al., 2008) were used. In the test by the NTP, where tests were performed in two laboratories, clearly induced SCE, with and without S-9 mix, was observed in one laboratory while in the other laboratory a positive response with S-9 mix only was reported. In the study by Speit et al., (2008) SCE were induced by glutaraldehyde in V79 cells in a concentration-dependent manner. The increase in SCE frequencies was small but reproducible and statistically significant. In an unscheduled DNA Synthesis study by St. Clair et al. (1991), rat hepatocytes were exposed to 0.1, 0.5, 1, 5, 10, 50 and 100 µM glutaraldehyde without metabolic activation. At concentrations exceeding 100 µM signs of toxicity became evident. At both highest tested concentrations of 50 and 100 µM glutaraldehyde a positive result was obtained. In a Bacillus subtilis recombination assay (Sakagami, 1988), glutaraldehyde was tested up to a concentration of 300 µg/mL in the presence and absence of S9 -mix. Both in the presence and in absence of S9 mix, glutaraldehyde was found to be positive in DNA-damaging capacity. In an Umu test (Sakagami, 1988) glutaraldehyde was tested in S. typhimurium TA 1535 pSK1002 with and without S9 -mix at concentrations of 1 - 100 µg/mL. Glutaraldehyde showed positive genotoxicity independent of metabolic activation system.

In vivo

Micronucleus assay

In a micronucleus assay, male and female CD-1 mice were exposed to the test substance via intraperitoneal injection at 0, 2, 10, and 20 mg/kg bw and sacrificed 24, 48 and 72 hours later and subsequently femoral bone marrow was examined for polychromatic erythrocytes (PCEs), micronucleated polychromatic erythrocytes (MPCEs) and for normochromatic erythrocytes /NCEs). (Pharmakon 1994). The test was performed according to guideline EPA OPP 84 -2 and following GLP. Only in females exposed to 10 mg/kg bw and sacrificed at time point 24 hours a significant increase in the frequency of micronucleated was observed. Therefore a confirmatory test was performed. In the confirmatory test, the statistically significant findings of the micronucleus test could neither be reproduced nor confirmed. Thus, it was concluded that glutaraldehyde was non-clastogenic in the in vivo micronucleus test.

Vergnes et al. (2002) exposed male and female Swiss Webster mice to glutaraldehyde (40, 80, and 125 mg/kg bw) via oral gavage and blood samples were collected after 30, 48, and 72 hours following dosage. The test was performed according to OECD guideline 474. No increase in micronucleated PCEs at sampling times of 30, 48 and 72 h following treatment with glutaraldehyde could be evidenced, indicating absence of an in vivo clastogenic potential of glutaraldehyde.

Chromosome aberration test

In a study by Microbiological Associates inc. (1987), a chromosome aberration test was performed similar to OECD guideline 475 and in compliance with GLP. Male and female Spraque-Dawley rats were exposed via oral gavage to the test substance for 1 day or five consecutive days. Single dosing was ca. 22.4, 74.6 and 224 mg/kg bw in males and ca. 16.3, 54.2 and 163 mg/kg bw in females and animals were sacrificed 8 or 12 hours after dosing and bone marrow cells were investigated. Repeated dosing was ca. 3.1, 10.5 and 31 mg/kg bw/day in males and ca. 2.5, 7.7 and 25 mg/kg bw/day in females and animals were sacrificed 12 hours after the last dose. No significant increase in number of aberrations/cell was seen in the treated groups and there was no evidence of a dose response at any sacrifice time point.

Vergnes et al. (2002) exposed male and female Sprague-Dawley rats to glutaraldehyde (Males: 12.5, 30 and 60 mg/kg bw, Females: 7.5, 20 and 40 mg/kg bw) via oral gavage and the rats were sacrificed after 12, 24 and 48 h following dosage and bone marrow cells were investigated. It was concluded that glutaraldehyde did not induce chromosomal aberrations in bone marrow cells of male and female rats.

Alkaline elution assay

The Procter and Gamble Company (1987) performed an in vivo alkaline elution assay for the induction of cross links in testicular cell DNA following oral gavage dosage of male Sprague-Dawley rats. Rats were exposed to 20 and 60 mg/kg bw and sacrificed after 2, 6, or 24 hours or exposed for five consecutive days and sacrificed two hours after the last application. In both tests, none of the X-irradiated samples from treated animals showed significant decrease in the rate of DNA elution indicative of cross links. It was concluded that glutaraldehyde did not induce cross-links in rat testicular DNA after in vivo single and repeated oral treatment.

Drosophila SLRL test

In a study by the NTP (1993) male Drosophila melanogaster were exposed in three tests to 0, 3000, and 4000 ppm via single injection, to 0 or 3500 ppm by oral feed over the larval development, or to 0, 7500, or 10000 ppm by oral feed over 3 days. For all 3 testing approaches, the percentage of sex-linked lethal mutations was within control range. Therefore it was concluded that glutaraldehyde was non-mutagenic to germ cells in the Drosophila SLRL test.

UDS

In an unscheduled DNA synthesis (UDS) test with mammalian liver cells (OECD 486 and GLP), male Wistar rats were exposed to glutaraldehyde (200 and 400 mg/kg bw) by a single oral gavage (BASF, 2002). Animals were sacrificed 3 or 14 hours after dose administration and hepatocytes were isolated from the liver. Evident signs of toxicity were seen in animals treated with the test material at both test doses and included piloerection, squatting posture and bloody snout. The microscopical evaluation and quantification of UDS showed that the results obtained for the treated animals were within the range of negative controls.

A UDS was also performed by Mirsalis et al. (1989). Male Fischer 344 rats were exposed to 30, 150 and 600 mg/kg bw via a single oral gavage. Two and 12 hours after exposure animals were sacrificed and hepatocytes were examined. In this test, glutaraldehyde failed to induce UDS in rats under in vivo test conditions.

Conclusion

Under in vitro conditions, glutaraldehyde is mutagenic to bacteria and to mammalian cells with and without metabolic activation, and is clastogenic in mammalian cells. However under in vivo conditions, glutaraldehyde was neither mutagenic nor clastogenic, and furthermore there were no indications of a mutagenic potential in germ cells. As all in vivo tests were negative, glutaraldehyde does not meet the criteria for classification as a mutagen, and no genotoxicity has to be expected under normal use conditions. The negative results obtained from in vivo testing of glutaraldehyde probably are related to the low bioavailability of the chemical in the body, its rapid elimination either via metabolic pathways or via binding to macromolecules, and its reactivity with tissue proteins. Because of these factors, only limited contact of glutaraldehyde with the intracellular genetic material has to be expected.

Short description of key information:
A series of valid in vitro and in vivo genotoxicity tests is available. The in vitro testing of glutaraldehyde indicate that glutaraldehyde is mutagenic to bacteria, and clastogenic and mutagenic to mammalian cells. However, all performed in vivo test indicate that glutaraldehyde is neither mutaganic nor clastogenic in vivo. Overall, it can be concluded that glutaraldehyde does not have to be classified for genetic toxicity.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Under in vitro conditions, glutaraldehyde is mutagenic to bacteria and to mammalian cells in culture with and without metabolic activation, and is clastogenic in mammalian cells. However under in vivo conditions, glutaraldehyde was neither mutagenic nor clastogenic, and furthermore there were no indications of a mutagenic potential in germ cells. Therefore, no classification is warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.