C,C'-azodi(formamide)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three Ames test according to OECD guidelines and GLP principles, both indicating mutagenic properties with and without metabolic activation. A positive result for mutagenicity in bacteria was also reported in a publication (Hachiya, 1987).

A chromosome aberration study was performed according to OECD guideline 473 and GLP principles, which indicated clastogenic properties of ADCA, in the presence and absence of metabolic activation.

A CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay was performed comparable to OECD guideline 476, in this assay no dose-dependent increases in the mutant frequencies of the cultures treated with the test article with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

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 methodology was broadly consistent with modern OECD test guideline 476, and a claim of GLP compliance was made, however as the guideline itself was not directly followed, and as the study report is missing a few very important details (test substance purity, for example), the study cannot be considered reliable without restrictions.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

: No lot number, purity or expiry date was indicated.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cell culture: Chinese hamster ovary cells, clone K1, subclone BH4, designated CHO-K1-BH4, from Dr. AbrahamW. Hsie, Biology Division, Oak Ridge National Laboratories, PO Box Y, Oak Ridge, Tennessee 37380
- Type and identity of media: F12FCM5 (Ham's F12 supplemented with 5% dialyzed and heat-inactivated bovine serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors

Test concentrations with justification for top dose:

cytotoxicity: 0.01,0.04, 0.13, 0.4, 1.3, 4.0, 13.3, 40, 133 and 400 µg/ml
mutation assay: 5, 16.7, 50, 167 and 500 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: solubility

All required dilutions were made with DMSO, Lot #741643, supplied by Fisher Scientific.
Dilutions were prepared the day of the test. Dosing solutions were used within two hours of preparation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: ethyl methanesulfonate [EMS, 200 ug/ml (Sigma M0880)] with S9: dimethylnitrosamine [DMN, 100 ug/ml (Aldrich N2,500-1)]

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period:none
- Exposure duration: 19h
- Expression time (cells in growth medium): 8days

NUMBER OF REPLICATIONS:5

DETERMINATION OF CYTOTOXICITY
- Method: relative cell survival

Evaluation criteria:

The mutant frequency, expressed as TGr mutants/10^6 clonable cells, was calculated by dividing the total number of mutant clones by the number of cells plated, corrected for the cloning efficiency (average of three plates) of the cells at the time of mutant selection.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Celogen AZ, was evaluated in a cytotoxicity prescreen with [2% (v/v)] and without S-9 at dose levels of 0.01, 0.04, 0.13, 0.4, 1.3, 4, 13.3, 40, 133 and 400 µg/ml. The 400 µg/ml dose approached the limit of solubility of the test article. The doses evaluated produced no detectable cytotoxicity to the CHO cell line without metabolic activation and produced 63.1% relative cell survival with metabolic activation at the 400 µg/ml level.
Based upon these findings, Celogen AZ, was evaluated in duplicate cultures in the mutation assay at dose levels of 5, 16.7, 50, 167 and 500 µg/ml with and without S-9.
There were no dose-dependent increases in the mutant frequencies of the cultures treated with the test article. The 500 µg/ml dose approximated the limit of solubility of the test article in DMSO. Therefore, the test article did not exhibit mutagenic activity under the conditions of the assay. This study fulfilled all the criteria of an acceptable assay.

Mutagenicity Data
Control Cultures
compound	µg/ml	S-9	Relative Initial Survival (%)	Total No. of Mutants (5 plates)	Cloning Efficiency (%)	Mutant Frequency (Mutants/10^6 clonable cells)
Untreated		(-)	108.2	1	68.8	1.5
Untreated		(-)	91.8	0	60.3	0.0
Untreated		(+)	90.0	0	74.3	0.0 a
Untreated		(+)	91	0	71. 7	0.0 a
DMSO	10	(-)	118.2	1	82.2	1.2
DMSO	10	(-)	86.2	1	72.3	1.4
DMSO	10	(+)	82	2	65.2	3.1
DMSO	10	(+)	98.9	8	50.3	15.9
EMS	200	(-)	88.4	175	57.5	304.3
EMS	200	(-)	82.7	149	43.3	344.1
DMN	100	(+)	12.5	90	30.6	294.1
DMN	100	(+)	10.9	74	41.2	179.6
Celogen AZ	5	(-)	99	1	57.8	1.7
Celogen AZ	5	(-)	105.6	1	75.3	1.3
Celogen AZ	16.7	(-)	82.4	1	63.2	1.6
Celogen AZ	16.7	(-)	64.9	2	57.5	3.5
Celogen AZ	50	(-)	88.8	1	58.5	1.7
Celogen AZ	50	(-)	83.2	0	46.3	0
Celogen AZ	167	(-)	109.1	0	64.3	0.0 a
Celogen AZ	167	(-)	76.1	0	58.7	0.0 a
Celogen AZ	500	(-)	48.7	1	80.7	1.2
Celogen AZ	500	(-)	54.5	10	65.2	15.3
Celogen AZ	5	(+)	86.6	1	56.5	1.8
Celogen AZ	5	(+)	74.2	1	60.2	1.7
Celogen AZ	16.7	(+)	96.8	0	53	0
Celogen AZ	16.7	(+)	116.4	1	54	1.8
Celogen AZ	50	(+)	92	1	65.5	1.5
Celogen AZ	50	(+)	90.4	2	34.8	5.7
Celogen AZ	167	(+)	22.7	1	64.2	1.6
Celogen AZ	167	(+)	26	1	64.2	1.6
Celogen AZ	500	(+)	7.5	0	70.8	0.0 a
Celogen AZ	500	(+)	5.7	0	65.2	0.0 a
a = No scorable mutants

Conclusions:

Celogen AZ did not exhibit mutagenic activity under the conditions of the assay.

Executive summary:

Celogen AZ, was evaluated in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay to determine its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in cultured Chinese hamster ovary (CHO) cells (Pharmakon Research International 1984, PH314 -UN-003 -84). Cytotoxicity of the test article was first estimated in a prescreen by exposing CHO cells to 10 levels of Celogen AZ in the presence and absence of metabolic activation. The metabolic activation mixture (S-9) contained 2% (v/v) of an Aroclor 1254-induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors. Doses of 0.01, 0.04, 0.13, 0.4, 1.3, 4, 13.3, 40, 133 and 400 µg/ml were evaluated. The 400 µg/ml dose approached the limit of solubility of the test article. The doses evaluated produced no detectable cytotoxicity to the cell line without metabolic activation and resulted in 63.1% relative cell survival with metabolic activation at the 400 µg/ml dose.

Based upon these findings, Celogen AZ was evaluated in duplicate cultures in the mutation assay at dose levels of 5, 16.7, 50, 167 and 500 µg/ml with and without S-9. The 500 µg/ml dose approximated the limit of solubility of the test article in DMSO.

There were no dose-dependent increases in the mutant frequencies of the cultures treated with the test article. Therefore, the test article did not exhibit mutagenic activity under the conditions of the assay. This study fulfilled all the criteria of an acceptable assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 March - 14 October 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Hams F12 medium (Imperial, supplemented with 5% foetal calf serum (Gibco) at 37°C in a humid atmosphere containing 5% carbon dioxide in 175 cm^2 plastic tissue culture flasks (Nunc).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

300, 150, and 30 µg/ml in the absence of metabolic activation when treated for 21 hours,
300, 200, and 3µg/ml in the presence of metabolic activation, 21 hour harvest,
260, 130 and 26 µg/ml in the absence of metabolic activation when treated for 45 hours,
24, 16 and 3.2 µg/ml in the presence of metabolic activation, 45 hour harvest.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C, 0.4 and 0.1 µg/ml: in absence of S9 (batch 96F-0547-1), Cyclophosphamide, 20 µg/ml, in presence of S9 (batch 123F-0283); Carbendazim for polyploidy, 12.5, 10 and 5 µg/ml, (batch D2150211)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: doubling time of CHO cells is 13 hours
- Exposure duration: 21, 4, 45 and 4 hours at 37°C
- Selection time (if incubation with a selection agent):Two hours before the end of the 21 or 45 hour incubation period.
- Fixation time (start of exposure up to fixation or harvest of cells):

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.25µl/ml
STAIN (for cytogenetic assays):10% Giemsa

NUMBER OF CELLS EVALUATED: Approximately 100 metaphase figures were examined where possible from each culture, with normally a maximum of 25 from each slide.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; metaphase

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

equivocal results at highest dose, positive effect on polypoid cell at highest dose, 45 hour treatment and harvest without S9

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

4 hour treatment, harvest at 45 hours

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Both clastogenic positive control compounds, mitomycin C (0.4 and 0.1 µg/ml) and cyclophosphamide (20 µg/ml), caused statistically highly significant increases in the proportion of metaphase figures containing aberrations when compared with the relevant solvent controls. The positive control compound for polyploidy, carbendazim, (12.5, 10 and 5 µg/ml) caused statistically highly significant increases in the proportion of polyploid cells (i.e. cells with 27 or more chromosomes).

In the absence of metabolic activation when treated for 21 hours, Unifoam AZ SO-NL caused a just statistically significant (P<0.05) increase in the proportion of cells with chromosomal aberrations, at the highest concentration, 300 µg/ml, when compared with the solvent control.
Therefore, unifoam AZ SO-NL has shown an equivocal response to the test for determining clastogenic activity. There was no increase in the number of polyploid cells when compared with the solvent control, at any dose level of unifoam AZ SO-NL, in this treatment regime.

In the presence of metabolic activation, 21 hour harvest, statistically highly significant increases (P<0.001) in the proportion of cells with chromosomal aberrations were observed at the lowest concentration, 3 µg/ml of Unifoam AZ SO-NL only. However, the toxicity profile observed for this treatment set was unusual, with no direct relationship between concentration of test compound and toxicity to the cells. Clear toxicity was seen at lower levels of test compound while at higher levels the toxicity became less apparent. This effect was observed in several experiments. The pattern of clastogenicity observed fits the toxicity profile directly (i.e. the higher levels of chromosome damage were observed at the lower concentrations of Unifoam AZ SO-NL). Therefore, it can be concluded that unifoam AZ SO-NL has shown clear evidence of clastogenic activity in this treatment set. No statistically significant increases in the proportion of polyploid cells were observed at any concentration of the test compound.

In the absence of metabolic activation when treated for 45 hours, unifoam AZ SO-NL caused a just statistically significant (P<0.05) increase in the proportion of metaphase figures containing chromosomal aberrations, including gaps at the highest concentration 260 µg/ml only. This increase, however, falls within the historical control range of this laboratory and is not considered to be indicative of clastogenic activity. Unifoam AZ SO-NL did, however, cause a statistically significant (P<0.001) increase in the proportion of polyploid cells at the highest concentration, 260 µg/ml. Although this increase is statistically significant, it falls within the historical control range of this laboratory and is not considered to be clearly indicative of polyploidy-inducing activity.

In the presence of metabolic activation, 45 hour harvest, unifoam AZ SO-NL caused a statistically significant increase in the number of aberrant cells, P<0.05, excluding gap damage and P<0.01 including gap damage and at the highest dose level, 24 µg/ml and P<0.05 excluding gap damage at 16 µg/ml, when compared with the solvent control. No statistically significant increases were observed at the lowest dose level therefore producing an equivocal response for the test to determine the clastogenic activity of the test compound. Statistically significant increases in the proportion of polyploid
cells, when compared with the solvent control, can be seen at 16 µg/ml (P<0.05) and 24 µg/ml (P<0.01) of Unifoam AZ SO-NL, indicating evidence of polyploidy-inducing activity.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

In the presence of metabolic activation, unifoam AZ SO-NL has shown clear evidence of clastogenic activity when harvested 21 hours after the addition of test compound and has shown evidence of polyploidy-inducing activity when harvested 45 hours after addition of test compound. An equivocal response to the test for determining clastogenic activity was obtained in the absence of metabolic activation when treated for 21 hours and in the presence of metabolic activation when harvested 45 hours after the addition of test compound. An equivocal response was also obtained for the test to determine the polyploidy-inducing activity of the compound, in the absence of metabolic activation when treated for 45 hours.

Executive summary:

A chromosome aberration assay was performed to determine the potential of the test substance Unifoam AZ SO-NL to cause chromosomal aberrations or polyploidy in mammalian cells (HLS 1989, OCI78a/881528). The study was conducted according to OECD and Japanese test guidelines, and in compliance with GLP.

Chinese Hamster Ovary cells were incubated with the test compound both with and without supplementary metabolic activation (rat S-9 mix). Two treatment periods were used in the absence of metabolic activation: 21 and 45 hour treatments which were both harvested immediately at the end of the treatment period. Two treatment periods were used in the presence of S-9 mix: 4 hour treatment, harvested 17 hours later, and 4 hour treatment, harvested 41 hours later.

The 21 hour test in the absence of metabolic activation produced an equivocal response to the test for determining clastogenic activity, but no significant increase in the proportion of polyploid cells was noticed. In the test with metabolic activation and a 21 hour harvest, a highly statistically relevant increase in the proportion of metaphase figures containing chromosomal aberrations was observed at 3 µg/mL of Unifoam AZ SO-NL only. The toxicity profile obtained for this treatment set was unusual with clear toxicity observed at lower concentrations of the test compound, and becoming less toxic at higher levels. The clastogenicity pattern fit directly with the toxicity pattern. This indicates clear evidence of clastogenic activity in this treatment regime. No statistically significant increases in the proportion of polyploid cells were observed at any concentration of the test compound. In the absence of metabolic activation when treated for 45 hours, the test substance caused a just statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations including gaps at one concentration, however the increase fell within the hostorical control range of the laboratory, and was not considered to be indicative of clastogenic activity. Similarly an increase in the proportion of polyploid cells was observed, but the increase fell within the histrical control range of this laboratory, and was not considered to be clearly indicative of polyploidy inducing activity. The 45 hours harvest test in the presence of metabolic activation produced an equivocal response for clastogenic activity, but a positive indication for polyploidy-inducing activity.

It was concluded that unifoam AZ SO-NL has shown evidence of both clastogenic and polyploidy-inducing activity in this in vitro cytogenetic test system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 April - 14 May 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon (S. typhimurium)
Tryptophan operon (E. coli)

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 from liver of rats treated with i/p injection of Aroclor 1254 (diluted in Arachis oil to 200 mg/ml) at a dosage of 500 mg/kg.

Test concentrations with justification for top dose:

5000, 500, 50, and 5 µg/plate (range finding). 5000, 2500, 625, 312.5 µg/plate (mutation test).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulphoxide
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: 2-aminoanthracene at 0.5 µg/plate (TA 1538, TA 98), at 1 µg/plate (TA 100), at 2 µg/plate (TA 1535, TA 1537), at 20 µg/plate (WP2 uvrA).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

With S9: at 3 µg/plate (TA 100), at 2 µg/plate (WP2 uvrA), at 5 µg/plate (TA 1535)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

With S9: at 80 µg/plate with TA 1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

With S9: at 1 µg/plate(TA 98), at 2 µg/plate (TA 1538)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Preincubation period: None
- Exposure duration: 72 hours at 37°C

SELECTION AGENT (mutation assays): histidine or tryptophan

NUMBER OF REPLICATIONS: three at each dose level

NUMBER OF CELLS EVALUATED: 0.1 ml = 2x10^9 cells/ml

DETERMINATION OF CYTOTOXICITY
- Method: substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group.
A compound is deemed to provide evidence of mutagenic potential if a statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In both mutation tests, highly significant dose-related increases in revertant colony numbers were observed with tester strains TA 1535 and TA 100, particularly in the presence of metabolic activation, following treatment with Unifoam AZ SO-NL.

Conclusions:

It is concluded that Unifoam AZ SO-NL shows clear evidence of mutagenic activity when tested in this bacterial system.

Executive summary:

A bacterial reverse mutation assay was performed to determine the potential of the test substance Unifoam AZ SO-NL to cause gene mutation (HLS 1988, OCI77/88757). The study was conducted in accordance with official test guidelines, and in compliance with GLP.

Five mutant strains of Salmonella Typhimurium (TA100, TA 1535, TA 98, TA 1537, and TA 1538) and one mutant strain of Escherichia coli (WP2 uvrA) were exposed to the test substance by plate incorporation. The test was performed both in the presence and in the absence of metabolic activation, and both negative (solvent) and relevant positive controls were used for each strain. A preliminary test was performed, and then a main mutation test was performed.

In both mutation tests, highly significant dose-related increases in revertant colony numbers were observed with tester strains TA 1535 and TA 100, particularly in the presence of metabolic activation, following treatment with Unifoam AZ SO-NL.

It is concluded that Unifoam AZ SO-NL shows clear evidence of mutagenic activity when tested in this bacterial system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Three in vivo mouse micronucleus studies were performed, two of these were done comparable to OECD guidance and under GLP. All three studies resulted in negative outcome.

An in vivo Comet assay was performed according to the OECD guideline and following GLP principles, the results indicates that ADCA does not induce DNA damage in vivo.  

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River U.K. Limited, Margate, Kent, England
- Age at study initiation:35 days old
- Weight at study initiation:22 and 24 grams
- Assigned to test groups randomly: yes
- Fasting period before study: The animals were deprvied of diet overnight prior to and for two hours after oral dosing.
- Housing:Each group of 2 or 5 mice was kept in a plastic disposable cage
- Diet (e.g. ad libitum):free access to pelleted Labsure LAD 1 rodent breeding diet.
- Water (e.g. ad libitum):free access to tap water.
- Acclimation period:4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22
- Humidity (%):no data
- Air changes (per hr):30
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: To: no data

Route of administration:

oral: gavage

Vehicle:

1% methylcellulose

Details on exposure:

Preliminary toxicity test:
Increasing dosages of Unifoam AZ SO-NL were administered up to a maximum of 5000 mg/kg bodyweight (0, 312.5, 625, 1250, 2500, 5000mg/kg). Twelve male and twelve female mice were used in this experiment (2mice/sex/group).
Following dosing, the animals were observed regularly during the working day for a period of 72 hours, and any mortalities or signs of malreaction during the experiment were recorded.

Duration of treatment / exposure:

single treatment

Frequency of treatment:

single treatment

Post exposure period:

24, 48, and 72 hours for Unifoam AZ SO-NL treated groups
24 hours for positive control group (Mitomycin C)

Remarks:

Doses / Concentrations:
5000mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

Vehicle comtrol group: 15 males and 15 females
Unifoam AZ SO-NL group: 19 males and 17 females
Mitomycin C positive control group: 5 males and 5 females

Control animals:

yes

Positive control(s):

Mitomycin C, obtained from Sigma London Chemical Company Limited (batch number 96F-0547-1) was tested at 12mg/kg.
It was prepared as a solution in sterile 0.9% saline at a concentration of 0.6 mg/ml.

Tissues and cell types examined:

Micronucleated cells

Details of tissue and slide preparation:

Following dosing, the animals were examined daily and weighted prior to dosing, and any mortalities or clinical signs of reaction to the test compounds were recorded. Five males and five females from the negative control and test compound groups were sacrificed 24, 48, and 72 hours after dosing. The positive control group was sacfrificed 24 hours after dosing. The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and one epiphysis removed from each bone.

A direct bone marrow smear was made onto a slide containing a drop of calf serum. One smear was made from each femur. The prepared smears were air-dried and stained for 10 minutes in 10% Giemsa. Following rinsing and differentiation in buffered distilled water for 10 minutes, the smears were air-dried and mounted with coverslips using DPX.

Stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal. The ratio of polychromatic to normochromatic erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated normochromatic erythrocytes was also kept.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

but limit comcentration tested

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 0-5000mg/kg
- Solubility: no data
- Clinical signs of toxicity in test animals:hunched posture and piloerection in the high dose groups until 4 hours after dosing

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):Unifoam AZ SO-NL did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes at any of the three kill times - (P>0.05 using Wilcoxon's sum of ranks test. Unifoam AZ SO-NL did not cause any substantial increases in the incidence of micronucleated normochromatic erythrocytes at any of the three kill times.
Mitomycin C caused large, highly significant increases (P<0.001) in the frequency of micronucleated polychromatic erythrocytes.
- Ratio of PCE/NCE (for Micronucleus assay):Unifoam AZ SO-NL failed to cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes (P>0.05 using Wilcoxon's sum of ranks test). Mitomycin C caused statistically significant decreases in the ratio (P<0.05).

Conclusions:

Negative

Executive summary:

In this assessment of the effect of Unifoam AZ SO-NL on the incidence of micronucleated polychromatic erythrocytes in mice, a dosage of 5000 mg/kg bodyweight was administered orally, by intragastric gavage (HLS 1988, OCI79/88802). (A preliminary toxicity test had been carried out to determine the toxicity of Unifoam AZ SO-NL). As no toxicity was observed at a dose level of 5000 mg/kg, the highest dose recommended for acute toxicity tests, this dosage was chosen for the main test.

Negative and positive control groups were dosed in an identical manner, orally by intragastric gavage. The negative control group received the vehicle, 1% methylcellulose. The positive control group was treated with mitomycin C, at 12 mg/kg.

Bone marrow smears were obtained from the negative control and test compound groups at 3 sampling times; these being 24, 48 or 72 hours after dosing. Bone marrow smears were obtained from the positive control group 24 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

 

At all sampling times, mice treated with Unifoam AZ SO-NL showed no significant increase in the frequency of micronucleated polychromatic erythrocytes. There was no significant decrease in the ratio of polychromatic to normochromatic erythrocytes after treatment of the animals with Unifoam AZ SO-NL. The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated polychromatic erythrocytes together with decreases in the ratio of polychromatic to normochromatic erythrocytes.

It is concluded from the results obtained that Unifoam AZ SO-NL shows no evidence of mutagenic potential or bone marrow cell toxicity when administered orally in this in vivo test procedure.

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:

24 March 2020 to 11 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was performed after final ECHA decision was received (Decision number: CCH-D-2114461492-49-01/F).

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- sex: dose-range finding study: male/female; main study: since there were no substantial differences in toxicity between sexes only males were used in the main study. 5 males / test group.
- Age at study initiation: 6 weeks
- Weight at study initiation: mean body weights were 179.4 ± 8.8 g and the range 159 – 193 g
- Assigned to test groups randomly: yes
- Fasting period before study: A limited quantity of food was supplied during the night before dosing (approximately 7 g/rat) until maximum 4 hours after administration of the test item.
- Housing: Polycarbonate cages (Makrolon MIV type; height 18 cm.) containing sterilized sawdust as bedding material equipped with water bottles.
During treatment in the dose-range finding study, polycarbonate cages containing sterilized sawdust as bedding material equipped with water bottles.
Up to 5 animals of the same sex and same dosing group together.
Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean temperature 20 to 21°C
- Humidity (%): mean relative humidity of 48 to 53%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

Route of administration:

oral: gavage

Vehicle:

1% carboxymethyl cellulose (CMC)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in 1% carboxymethyl cellulose (CMC).

Duration of treatment / exposure:

two consecutive days

Frequency of treatment:

once daily

Post exposure period:

None - Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia and tissues were isolated.

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

positive control EMS

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

Test item

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Test item

Dose / conc.:

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

Remarks:

Test item

No. of animals per sex per dose:

5 males (main study)

Control animals:

yes, concurrent vehicle

Positive control(s):

ethyl methanesulfonate (EMS)

Tissues and cell types examined:

Liver, Stomach, Duodenum.

The isolation method for duodenum and for glandular stomach is based on the method for stomach that was used in the JaCVAM Comet validation study. The isolation method was based on the publication of Hu et al (2002). For more details under 'Any other information on materials and methods incl. tables'.

Details of tissue and slide preparation:

Preparation of Slides:
To the cell suspension, melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added (ratio 10:140). The cells were mixed with the LMAgarose and
50 µL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 15 to 20 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

Lysis, Electrophoresis and Staining of the Slides:
The cells on the slides were overnight (approximately 16 h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20 minutes (duodenum and stomach) and 30 minutes (liver) at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 V/cm for stomach and duodenum and 0.9-1 Volt/cm for liver. The electrophoresis was performed for 20 minutes (duodenum and stomach) and 30 minutes (liver) under constant cooling (actual temperature 4.0 to 4.5 °C). Electrophoresis parameters used at CRL Den Bosch were optimized to obtain reproducible acceptable low background DNA damage values and clear responses of the positive control EMS. After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in Absolut ethanol (>=99.6%, Merck) and allowed to dry at room temperature. The slides were stained for 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

Sampling, fixation and storage of tissue for histotechnology and histopathology:
Part of the duodenum, stomach and liver from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was needed.

Evaluation criteria:

A test item is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided,
p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent
negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05)
increase in percentage Tail Intensity is detected compared with the concurrent negative
control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany)

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No biologically relevant dose related increase in the mean Tail Intensity (%) was observed in duodenum, stomach and liver cells of test item treated male treated animals compared to the vehicle treated animals. All Tail Intensity values were within the 95% control limits of the distribution of the historical negative control.
The mean Tail Intensity in duodenum, stomach and liver cells of vehicle-treated rats was 4.63 ± 0.68% (mean ± SD), 4.51 ± 1.50% (mean ± SD) and 2.87 ± 0.40% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 37 ± 1.84% (mean ± SD), 58 ± 6.96% (mean ± SD) and 85 ± 1.00% (mean ± SD) in male animals in duodenum, stomach and liver cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analysed and the highest test dose was the maximum dose required by the guidelines. Hence, all criteria for an acceptable assay were met.

There was no proof of internal exposure to the test item in the present study. Bioanalysis in blood was not performed and there was no indirect proof of exposure (no toxicity) in the present study. Since there was no proof of internal exposure there is no proof that the liver cells were exposed to the test item. On the other hand, the stomach and duodenum are first site of contact organs and were exposed to the test item.
Furthermore, the significant toxicity observed in the rabbit prenatal study (summarized in Section 7.8) indicates uptake of the test item after oral exposure.

Table 1: Overview Tail Intensity in Duodenum Cells of Male Rats

	Tail Intensity (%)	S.D.
0 mg/kg	4.63	0.68
500 mg/kg	5.10	1.16
1000 mg/kg	5.20	1.05
2000 mg/kg	5.61	1.45
EMS 200 mg/kg	36.93	1.84

 

Table 2: Overview Tail Intensity in Stomach Cells of Male Rats

	Tail Intensity (%)	S.D.
0 mg/kg	4.51	1.50
500 mg/kg	4.64	0.91
1000 mg/kg	5.40	1.44
2000 mg/kg	4.91	1.47
EMS 200 mg/kg	57.66	6.96

 

Table 3: Overview Tail Intensity in Liver Cells of Male Rats

	Tail Intensity (%)	S.D.
0 mg/kg	2.87	0.40
500 mg/kg	3.35	0.19
1000 mg/kg	3.08	0.24
2000 mg/kg	3.59	0.36
EMS 200 mg/kg	85.25	1.00

 

 

Table 4: Historical data Comet assay Negative control

	Liver Tail Intensity (%) Males and Females	Duodenum Tail Intensity (%) Males and Females	Glandular Stomach Tail Intensity (%) Males and Females
Mean	1.96	3.06	2.45
SD	0.92	1.52	1.39
n	85	45	60
Lower control limit (95% control limits)	0.27	-0.86	-1.07
Upper control limit (95% control limits)	3.65	6.97	5.96

SD = Standard deviation

n = Number of observations

Liver, Stomach, Duodenum:

Historical control data from experiments performed in Jan 2018 – July 2019

 

Table 5: Historical data Comet assay (200 mg/kg EMS orally dosed for two consecutive days)

	Liver Tail Intensity (%) Males and Females	Duodenum Tail Intensity (%) Males and Females	Glandular Stomach Tail Intensity (%) Males and Females
Mean	89.53	41.17	55.16
SD	6.89	14.03	14.23
n	80	44	59
Lower control limit (95% control limits)	79.70	20.78	34.74
Upper control limit (95% control limits)	99.36	61.56	78.58

SD = Standard deviation

n = Number of observations

Liver, Stomach, Duodenum:

Historical control data from experiments performed in Jan 2018 – July 2019

 

 

Conclusions:

An in vivo Comet assay was performed according to the OECD guideline and following GLP principles. Based on the results it is concluded that the test item does not cause DNA damage in duodenum, stomach and liver cells sampled approximately 3-4 hours post dosing, in male rats that were dosed via oral gavage for two consecutive days up to a dose of 2000 mg/kg (the recommended highest dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A mouse micronucleus study was performed in which mice were orally dosed up to 5000 mg/kg bw.  In a second mouse micronucleus study, mice were dosed i.p at 150 mg/kg bw (maximum dose related to mortality at higher dose levels observed in a dose range finding study). Exposure of the bone marrow was confirmed in this study. In both studies no statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was observed. A negative result in an in vivo Micronucleus study was also reported in a publication (Hachiya, 1987).

An in vivo Comet assay was performed according to the OECD guideline and following GLP principles, in which rats were orally dosed up to and including the limit dose of 2000 mg/kg bw. The results did not indicate DNA damage in liver cells and at first point of contact (duodenum and glandular stomach). Although systemic exposure to ADCA could not be concluded in this study, the significant toxicity after oral exposure observed in the rabbit prenatal study (summarized in Section 7.8) indicates that ADCA becomes systemically available after oral dosing. 

The two methods (in vivo Micronucleus and in vivo Comet assay) assess different mode of actions. Based on the negative results in the studies it is concluded that ADCA does not induce chromosome aberrations and/or DNA damage (eg point mutations) in vivo.

The current data-set is sufficient to conclude that ADCA is not mutagenic in vivo.

Justification for classification or non-classification

Based on the current data-set, ADCA is not classified for genetic toxicity according to Regulation (EC) No 1272/2008.