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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1982
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: The study did not use negative or positive controls, only two concentrations of the chemical were tested. No metabolic activation system was used. The contact time between chemical and cells was insufficient.

Data source

Reference
Reference Type:
publication
Title:
Differential sensitivity of muntjac lymphocyte chromosomes to mitocyin C, bromodeoxyuridine and hydroxylamine at different cell cycles
Author:
Gupta P., Sharma T.
Year:
1982
Bibliographic source:
Mutation Research 98:161-174

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No controls were used, only two concentrations of the test chemical were tested, no metabolic activation system was used.
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
Hydroxylammonium hydrochloride
IUPAC Name:
Hydroxylammonium hydrochloride
Test material form:
not specified
Details on test material:
No details stated

Method

Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Peripheral blood lympocytes from a male muntjac (Muntiacus muntjak)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Metabolic activation system:
Not applicable
Test concentrations with justification for top dose:
25, 50 µg/ml
Vehicle / solvent:
Cell medium
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Medium
True negative controls:
no
Positive controls:
no
Positive control substance:
no
Details on test system and experimental conditions:
Peripheral blood lymphocytes obtained from a male muntjac (Muntiacus muntjak) were cultured in medium containing Parker 199 (15ml), phytohaemmagglutinin (0.1 ml) and human AB serum (20% heat-inactivated). The cultures were grown at 37C for 32 hours, when the first cycle metaphases were obtained. Hydroxylammonium hydrochloride was added to the cells during each cell cycle. The timings for each cell cycle were obtained autoradiographically and were as follows:
G1, 1 hour after initiation of the cultures;
Early S, 18.5 hours after initiation of the cultures;
Late S, 4.5 hours before fixation of the cultures;
G2, 2 hours before fixation of the cultures.

The chemical fixation time was carried out at 32 hours (some additional time was allowed for mitotic delay due to the presence of hydroxylammonium hydrochloride.

The growing cells were pulse treated for 1 hour dugin G1, early S, late S and G2 stages with medium containing 25 and 50 ug/ml of hydroxylamine hydrochloride. After treatment cells were washed twice with chemical free medium and re-incubated with the conditioned medium. Colcemid was added 2 hours before cell harvesting, following by treatment with hypotonic solution (0.56% KCl) and subsequent fixation by treatment with methanol/acetic acid fixative (3:1). Slides were prepared by a flame-dryng technique and stained with Giemsa.
Evaluation criteria:
The slides containing the chromosomes were scored for the frequencies of induced aberrations. Aberrations observed included chromatid gaps, isochromatid breaks, interchanges, sister-chromatid union, translocations and dicentrics.
Statistics:
For each dose of the chemical tested, three replicates were performed. For each treatment, 2 replicate cultures were set up.

To find out whether the distribution of chromosomal aberrations were at random, expected values of chromosomal aberrations were calculated assuming that each chromosome would participate in aberration in proportion to its relative length.

Results and discussion

Test results
Species / strain:
lymphocytes: Peripheral blood lymphocytes from male Munjac (Muntiacus muntjak)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
50 µg/ml
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable

Any other information on results incl. tables

The authors observed cyclic variation in the sensitivity of muntjac lymphocytes to hydoxylammonium hydrochloride. The sensitivity of cells to hydroxylamine hydrochloride were found to increase as the cells moved through the cell cycle.

At 25 µg/ml, the frequencies of aberrations at late S ang G2 stages were almost equal. At 50 µg/ml the late S cells exhibited comparatively more aberration than G2 cells.

The aberrations observed were mainly constriction or gap types and were found more frequently in cultures exposed to hydroxylammonium hydrochloride during late S and G2 stages

Frequency of various morphological types of aberration on muntjac chromosomes induced by treatment with hydroxylammonium hydrochloride during different stages of the cell cycle (data was pooled from 3 independent experiments).

Treatment Stage

Concentration (µg/ml)

Total cells observed

Gaps

Chromatid breaks

Isochromatid breaks

Centromeric breaks

Secondary constriction breaks

Fragments

Dicentrices

Translocations

Chromatid exchanges

SU

G1

25

894

33

2

12

12

4

1

-

1

-

-

Control

400

4

3

1

-

-

-

-

-

-

-

50

983

47

7

8

2

5

1

1

1

-

1

Control

350

3

5

-

-

-

-

-

-

-

1

ES

25

753

39

2

11

4

13

2

-

1

1

2

Control

390

6

5

-

-

-

-

-

-

-

-

50

900

63

3

17

4

3

1

-

-

1

2

Control

275

3

2

3

-

-

-

-

-

-

-

LS

25

802

61

13

6

4

4

3

-

2

3

1

Control

400

6

3

1

-

-

-

-

-

-

-

50

744

127

11

33

3

8

1

-

-

3

3

Control

200

3

4

-

3

8

1

-

-

-

-

G2

25

861

71

30

9

14

14

5

-

1

-

2

Control

270

5

3

-

1

1

-

-

-

7

-

50

870

89

8

16

3

3

2

1

-

1

2

Control

350

6

4

-

-

-

-

-

-

-

-

The distribution of aberrations and test for randomness of chromosomal aberrations based on unit length of chromosomes after treatment with hydroxylammonium hydrochloride (25, 50 µg/ml) in muntjac lymphocytes. To determine whether the distribution of hydroxylammonium hydrochloride aberrations were random, the data were analysed statistically. The expected values were calculated by assuming that each chromosome would participate in aberration in proportion to its relative length.

 

Treatment stage

Concentration (µg/ml)

 

Chromosome Number

Total aberrations

Total cells observed

 

1

2

X

Y2

Y1

G1

25

Obs.

18

12

22

4

-

55

685

Exp.

18.04

12.8

12.5

8.5

2.1

55

 

50

Obs.

35

12

21

5

-

73

993

Exp.

23.9

17

18

11.3

2.8

73

 

ES

25

Obs.

24

13

28

11

-

76

906

Exp.

24.7

17.7

18.7

11.7

3.0

76

 

50

Obs.

48

16

26

4

-

94

900

Exp.

30.8

21.9

23.2

14.5

3.6

94

 

LS

25

Obs.

38

18

31

10

-

97

946

Exp.

31.8

22.6

23.9

15.0

3.7

97

 

50

Obs.

86

31

53

19

-

189

944

Exp.

61.9

44

46.6

29.2

7.3

189

 

G2

25

Obs.

59

32

46

9

-

146

894

Exp.

47

34

36

22.6

6.4

146

 

50

Obs.

49

28

35

6

-

128

870

Exp.

41

29.8

31.6

19.8

5.8

128

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation 50 µg/ml

Hydroxylammonium hydrochloride was found to increase the frequency of chromosome aberrations in the peripheral blood lymphocytes from male muntjac (Muntiacus muntjak) at concentrations of 50 ug/l. There was no increased frequency of chromosome aberrations at concentration of 25 µg/l. The study design did not use metabolic activation, furthermore the cells were only exposed to the chemical for a short period of time (1 hour), indicating that the result is likely to be result is insensitive.
Executive summary:

Hydroxylammonium hydrochloride was found to be cause chromosome aberrations in the peripheral blood lymphocytes from a male muntjac (Muntiacus muntjak). The study did not follow standardised guidelines, furthermore no controls or metabolic activation system was used in the study. Therefore although hydroxylammonium hydrochloride is considered genotoxic by this study design, the study design is not considered reliable.