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Gene mutation on bacteria

Two reverse mutation tests on bacteria (on S. typhimurium and on E. coli) are available and valid. Both tests have been realised according to protocol similar to the OECD guidelines. Results are homogenous and clearly indicate a positive effect of ADCA on S. typhimurium.

A third test also supports these findings, but is not sufficiently described.

 

The first bacterial reverse mutation assay was performed to determine the potential of ADCA (tested under the name of 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.

 

In the second test, ADCA (tested under the name of Celogen AZ-130) was evaluated for reverse gene mutation on bacteria (Ames test) in strains TA1535, TA1538, TA98, and TA100 of Salmonella typhimurium both with and without rat liver metabolic activation preparation at doses of 50, 166, 500, 1666, and 5000 µg/plate (Pharmakon Research International 1984, PH301 -UN-004 -84).

Strain TA1537 of Salmonella typhimurium was re-evaluated at a later date due to abnormal colony morphology in the original assay. Strain TA1535 was re-evaluated both with and without metabolic activation at dose levels of 500, 1000, 2500, 5000 and 7500 due to the positive results observed in the original assay.

Strain TA100 was also re-evaluated with metabolic activation preparation at dose levels of 500, 1000, 2500, 5000 and 7500 µg/plate to confirm an apparent dose-related response in the original assay.

Positive results were observed for test article, Celogen AZ-130, in strain TA1535 with and without metabolic activation at the 1666, 2500, 5000, and 7500 µg/plate levels. There was a dose-related increase in mutation frequency in strain TA100 with metabolic activation which reached values of 2.75 and 2.2 times the solvent control in the initial and repeat assays. All solvent and positive controls used in the evaluation of the test article were within the acceptable limits of mean historical data.

 

In the third test (not sufficiently described), in vitro assessment of the mutagenic potential of technical (production) azodicarbonamide, Celogen AZ 120, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) were exposed to the test material, diluted in dimethylsulphoxide which was also used as a negative control (HRC 1984, FSB144/84572 /2).

Two independent mutation tests were performed using agar plates, in the presence and absence of liver preparations from Aroclor 1254 induced rats.

In the preliminary dose range finding study no toxicity was observed up to the highest dose level used, 5000 µg/plate. Therefore a top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 3000,

1500, 1000, 500, 150, 50 µg/plate. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the metabolic activation system.

Evidence of mutagenic activity was seen in both the presence and absence of metabolic activation with tester strains TA 1535 and TA 100.

It is concluded that, when tested at dose levels up to 5000 µg/plate in DMSO, technical (production) azodicarbonamide, Celogen AZ 120 was strongly mutagenic in both the presence and absence of metabolic activation, possibly as a result of base-pair substitution.

 

 

In vitro chromosomal aberration test: One assay is available and valid.

A chromosome aberration assay was performed to determine the potential of ADCA (tested under the name of 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.

 

 

In vitro gene mutation on mammalian cells: One assay is available and valid.

ADCA (tested under the name of 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 pre-screen by exposing CHO cells to 10 levels of ADCA 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, ADCA 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.

 

Micronucleus test:

Two different studies have been realised, both according to protocols similar to international recognised guidelines.

In the first test, the effect of ADCA (tested under the name of Unifoam AZ SO-NL) on the incidence of mirconucleated polychromatic erythrocytes in the bone marrow of mice has been determined (HLS 1988, OCI79/88802). The study was conducted in accordance with an EC test guidelines and a draft OECD test guidelines, and was conducted in compliance with GLP.

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. (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.

 

Second test: In a preliminary dose-range-finding study, ADCA (tested under the name of Celogen AZ 130), was administered intraperitoneally to five groups (2 males and 2 females per group) of CD-l mice at dose levels of 50, 166.6, 500, 1666.6 and 3000 mg/kg of body weight (Pharmakon Research International 1984, PH309A-UN-003 -84). Signs were observed at all levels evaluated. All animals died at levels of 500 mg/kg and higher. No mortality was observed at the two lowest doses. Due to the severity of the signs and the mortality observed in· the study, 150 mg/kg was selected as the dose for the Micronucleus Test as an estimate of the maximum tolerated dose.

In the Micronucleus Test, three groups of ten animals (5 males and 5 females/group) were given single doses by intraperitoneal injection at 150 mg/kg and sacrificed at 30, 48 and 72 hours. Due to mortality observed in the 72 hour sacrifice group, additional animals were administered the test article to obtain sufficient animals for a reliable analysis to be made. Similar groups, serving as the positive (TEM) and negative control, Corn Oil, were evaluated concurrently.

Slides were prepared from the bone marrow of the femurs and stained. Coded slides were scored for the number of polychromatic erythrocytes (PCE) with micronuclei in 1000 PCE. The ratio of polychromatic to normochromatic erythrocytes per 1,000 erythrocytes was determined.

The results for Celogen AZ 130 were negative in the Micronucleus Test at a dose level of 150 mg/kg at all of the time intervals evaluated. These findings are based upon the inability of the test article to produce a statistically significant increase in the number of micronuclei in 1000 polychromatic erythrocytes in the treated versus the negative control group.


Short description of key information:
Gene mutation on bacteria - S. typhimurium, E. coli (in-vitro) - positive.
Chromosome aberration test - Chinese Hamster Ovary cells (in-vitro) - positive.
Gene mutation on mammalian cells - Chinese Hamster Ovary cells (in-vitro) - negative.
Micronucleus test - Mice (in-vivo) - negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Although the results of the in-vitro tests in bacteria (Ames tests) and in vitro chromosomal aberrations in mammalian cells were positive for mutation, an in vitro gene mutation study on mammalian cells showed a negative result, and two in-vivo micronucleus test in mouse are available and the result of both tests was negative; the overall conclusion for genetic toxicity is therefore that ADCA does not require classification as mutagenic. This conclusion is based on the criteria set out in EU directive 67/548/EEC (The Dangerous Substance Directive), and regulation 1272/2008 (The Classification, Labelling and Packaging Directive).