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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Ames tests: negative with and without s9; Mammalian cell gene mutation assay: negative with and without s9; in vitro micronucleus assay: negative with and without s9; in vivo micronucleus test: negative with and without s9.
Link to relevant study records
Reference
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
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Valid OECD 474 guideline study without deviations, performed under GLP
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
Species: NMRI mouse
Strain: Hoe:-NMRKf (SPF71)
Origin: HOECHST AG, Kastengrund, SPF breeding colony
Initial age at test: 07 - 12 weeks
Number of animals: 70 (35 males/35 females)
Body weights at start of study: males: average 31,3 g ( 27 - 37 g), females: average 24,5 g ( 22 - 28 g)
Acclimatization: at least 5 days
Food / water: rat diet Altromin 1324 (Altromin - GmbH, Lage/Lippe), ad libitum; tap water in plastic bottles, ad libitum
Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals.
Room temperature: 22 ±2 °C
Relative atmospheric humidity: 55 ±10 %
Lighting time: 12 hours daily
Animal identification: fur-marking with KMnO4 and cage numbering
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Preparation and administration of the test substance:
The test substance dilutions were freshly prepared each day. 2.500 mg sulfamic-acid was weighed into a beaker, mixed with deionized water, washed out into a 50 ml flask and topped up to the calibration mark with de-ionized water; a solution was formed. For the Endoxan stock solution, 5 ml distilled water were added to 100 mg Endoxan in an injection phial and shaken to form a clear solution. The daily solutions for administration were prepared from this stock solution. For this purpose, 2 ml of the 2 % stock solution were mixed with 6 ml distilled water. the substance was applied by gavage.
Duration of treatment / exposure:
24, 48 and 72 hours
Frequency of treatment:
single application
Post exposure period:
24, 48 and 72 hours per dose group (Positive control only 24 hours)
Remarks:
Doses / Concentrations:
0 and 200 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females per dose group and exposure period
Control animals:
yes, concurrent vehicle
Positive control(s):
5 males and 5 females dosed with 50 mg/kg bw Endoxan as positive control
Tissues and cell types examined:
Extraction of the bone marrow: The animals were sacrificed according to the test procedure 24, 48 or 72 hours after application by carbon dioxide asphyxiation. For each animal about 3 ml foetal bovine serum was poured into a silicone-coated centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed.
Details of tissue and slide preparation:
The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number, and air-dried for about 24 hours.
Staining:
- 3 minutes in May-Grunwalds solution
- 2 minutes in Hay-Grunwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts distilled water
- rinsing in distilled water
- drying
- providing with Entellan

Evaluation criteria:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition the ratio of polychromatic to normochromatic erythrocytes was determined. For evaluation all bone marrow smears are coded to ensure that the group to which they belonged remained unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occuring in 1000 counted polychromatic erythrocytes, and the number of normocytes with micronuclei occuring in 1000 counted normocytes, were evaluated statistically;
Statistics:
the method of binomial increase was used to show any increase as compared with the results of the controls (Miller R.G., Simultaneans Statistical Interference, Mc Graw-Hill, New York 1966). The results of the test substance at each killing time and dose results were compared with the corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Nemenyi (Brownlee, K.A., Statistical theory and methodology, Wiley, New York, 1965). The statistical evaluations were realized with the "Diamant" computer program SG-PA-4453, supplied by the Department Praktische Mathematik of Hoechst AG. All statistical results are based on the 95 % level of significance.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2 animals died within 24 hours
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Animals were treated with 0 or 200 mg sulfamic acid per kg body weight to study the induction of micronuclei in bone marrow cells of mice.
All animals survived after application of 0 or 200 mg per kg body weight. One animal died 90 minutes after application due to a failure of application per gavage. The following animals were also replaced due to a failure of application per gavage: Animal-No. 10, 16, 22, 37 and 39.
The following signs of toxicity were observed 24 hours after application: Abdominal position, back-arched position, tachypnoe, ptosis, piloerrection, hyperreflexia and change in gait.
The following signs of toxicity were observed 48 and 72 hours after treatment: Back-arched position, piloerrection, increased respiratory sounds, change in gait, abdominal position, tachypnoe and soft stools.
Animal-No. 13 and 48 died within 24 hours.
The dissection of the male animals revealed the following macroscopic findings: In the group treated with 200 mg sulfamic acid per kg body weight some animals had a distended urinary bladder filled with urine and the intestine filled with air or a yellowish content in the intestine.
At the dosage of 200 mg sulfamic acid per kg body weight and sacrificed 24 hours after application one animal showed light surface of the livers. Some females of the same treatment group showed 24, 48 and 72 hours after application yellowish content in the intestine, the stomach or the intestine filled with air. One animal had anaemic kidneys and livers.
The incidence of micronucleated polychromatic erythrocytes in each dose group of sulfamic acid was comparable with the data in the negative control groups. The number of erythrocytes with micronuclei did not differ significantly from the values of the simultaneous control animals for each of the three killing times investigated. This is true for the polychromatic erythrocytes as well as for the normocytes.
The ratio of polychromatic erythrocytes to normocytes remained uneffected by the test compound. Cyclophosphamid (Endoxan) induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females. The ratio of polychromatic erythrocytes to normocytes was not shifted.
Conclusions:
Interpretation of results (migrated information): negative
Under the conditions described the application of sulfamic acid did not lead to an elevated occurrence of micronuclei in the polychromatic erythrocytes.
It is concluded that sulfamic acid is not mutagenic in the micronucleus test.
Executive summary:

Sulfamic acid was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 200 mg sulfamic acid per kg body weight. The animals were treated once with the test substance and sacrificed according to the test procedure 24, 48 or 72 hours after administration of the test compound.

Endoxan was used as positive control substance at an oral dose of 50 mg per kg body weight. The incidence of micronucleated polychromatic erythrocytes was not effected by the test substance in comparison with the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased too.

The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with sulfamic acid. Endoxan (positive control) induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not shifted.

The results indicate that sulfamic-acid is not mutagenic in the micronucleus test under the conditions of the present study.

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

Additional information

Additional information from genetic toxicity in vivo:

Sulphamidic acid has been investigated in six in vitro studies and one in vivo study for mutagenicity and was found negative in all of them. Therefore, sulphamidic acid is considered non-mutagenic. The individual studies are summarized as follows:

De Flora et al. reported in 1981 and 1984 in two independent publications about investigations on mutagenicity of various compounds, amongst them sulphamidic acid, by using the Ames test system with salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 (published 1981) and with E. coli strains WP2, WP67 and CM871 (published in 1984). Sulphamidic acid was found negative (with and without metabolic activation) for mutagenicity in all strains tested.

Independently, Meyer et al., Hoechst AG, investigated sulphamidic acid in an Ames Study using salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and in addition E. coli WP2 uvr A bacteria (with and without metabolic activation) and could confirm the absence of mutagenic effects in this assay.

In an in vitro mammalian cell gene mutation test (S. Li 2010) according to OECD guideline 476 with Chinese hamster Ovary cells (with and without metabolic activation) no mutagenic potential of sulphamidic acid was found either and also in an OECD 476 study with Chinese Hamster Lung cells (HPRT locus of V79) no mutagenicity was found (with/without S9 activation, Bednáriková et al. 2010).

Finally, Beňo et al. in 2010 studied the effects of sulphamidic acid on human peripheral blood lymphocytes in a chromosome aberration study according to OECD 487 guideline (with and without metabolic activation) and could not detect any mutagenic effects.

The negative findings for mutagenicity from the six in vitro studies above were confirmed by Mayer et al. 1985 in an in vivo study according to OECD 474, performed under GLP. Under the conditions described the application of sulphamidic acid did not lead to an elevated occurrence of micronuclei in the polychromatic erythrocytes.

 

In conclusion all in vitro and in vivo mutagenicity studies showed no indication of mutagenic potential for sulphamidic acid and thus the substance is considered non-mutagenic.


Justification for selection of genetic toxicity endpoint
in vivo study, supported by in vitro studies

Justification for classification or non-classification

Based on the test results of six negative in vitro studies and one negative in vivo study, no classification for mutagenicity is required for sulphamidic acid according to regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 and in accordance with DSD (Directive 67/548/EEC).