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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
2000-06-15 to 2000-06-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
ZINN(II)-RICINOLEAT
IUPAC Name:
ZINN(II)-RICINOLEAT
Constituent 2
Reference substance name:
Reaction products of ricinoleic acid and linoleic acid and oleic acid with sodium hydroxide and tin (II) chloride
EC Number:
700-814-2
Molecular formula:
not applicable UVCB substance
IUPAC Name:
Reaction products of ricinoleic acid and linoleic acid and oleic acid with sodium hydroxide and tin (II) chloride
Test material form:
liquid: viscous

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 preparation from Aroclor 1254 induced rats
Test concentrations with justification for top dose:
3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
dimethyl sulfoxide
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: without S9: sodium azide, 9-aminoacridine, daunomycine, methylmethanesulfonate, 4-nitroquinoline N-oxide; with S9: 2-aminoanthracene.
Details on test system and experimental conditions:
Test System : Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale : Recommended test system in international guidelines (e.g. EPA, OECD, EEC).
Source : Dr. Bruce N. Ames, University of California at Berkeley, U.S.A. (Salmonella typhimurium strains); TA100 received 18-02-1993, used batches: T100.291 099 and TA100.040500; TA98 received 21-02-1991, used batch: T A98.170200; T A 1535 received 14-12-1994, used batch: T A 1535.170200; T A 1537 received 14-12-1994, used batch: T A 1537 .170200; Prof. Dr. B.A. Bridges, University of Sussex, Brighton, U.K., (Escherichia coli strain) WP2uvrA received 23-10-1987, used batch: EC.040500.
Preparation of
Bacterial cultures : Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking incubator (37 °C, 150 spm), until the cultures reached an optical density of 1.0 at 700 nm (10 9 cells/ml). Freshly grown cultures of each strain were used for a test.
Permeabilization of the
Escherichia coli strain : WP2uvrA bacteria were washed twice in 0.25 the original volume of ice-cold 0.12 M Tris-HCl buffer pH 8.0, then gently resuspended in 0.2 vol. 0.12 M Tris HCl, 0.5 mM EDTA pH 8.0, and shaken for 2.5 min at 37 °C. MgCI 2 was then added to a final concentration of
10 mM. The cells were centrifuged and resuspended in the original volume of nutrient broth.
Agar plates : Agar plates (0 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid, code l28) in Vogel-Bonner Medium E, 20 g glucose. N.B. The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin and 15 µg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan.
Top agar : Vogel-Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121°C.
Environmental conditions : All incubations were carried out in the dark at 37 °C. The temperature was monitored during the experiment.
Evaluation criteria:
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Experiment 1:
Precipitate: ZINN(II)-RICINOLEAT precipitated in the top agar at concentrations of 1000 and 3330 µg/plate. Precipitation of ZINN(II)-RICINOLEAT on the plates was observed at the start and at the end of the incubation period at the concentration of 3330 µg/plate.
Toxicity: In tester strain TA 1537 in the absence of S9-mix, a moderate reduction in the number of revertant colonies was observed at the test substance concentration of 1000 µg/plate. An extreme reduction in the number of revertant colonies and a slight reduction of the bacterial
background lawn were observed at the concentration of 3330 µg/plate. In the presence of S9-mix, no revertant colonies were observed at the test substance concentration of 3330 µg/plate. In tester strain TA 1535 and T A98, the bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.
Mutagenicity: No increase in the number of revertants was observed upon treatment with ZINN(II)-RICINOLEAT.
Experiment 2:
Precipitate: ZINN(II)-RICINOLEAT precipitated in the top agar at concentrations of 1000 µg/plate and upwards. Precipitation of ZINN(II)-RICINOLEAT on the plates was observed at the start and at the end of the incubation period at concentrations of 1670 and 3330 µg/plate.
Toxicity: In tester strain TA 100 in the absence and presence of S9-mix, a moderate reduction in the number of revertant colonies and a slight reduction of the bacterial background lawn were observed at the test substance concentration of 3330 µg/plate. In the other tester strains TA 1535, TA 1537, TA98 and WP2uvrA, the bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.
Mutagenicity: No increase in the number of revertants was observed upon treatment with ZINN(II)-RICINOLEAT.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1: MUTAGENIC RESPONSE OF ZINN(II)-RICINOLEAT IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND IN THE ESCHERICHIA COLI REVERSE MUTATION ASSAY

 

Dose [µg/plate]

Mean number of revertant colonies/3 replicates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

 

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

 

Without S9

Positive control

723±68

334±39

442±34

419±90

406±35

Solvent control

15±4

8±2

27±1

66±10

17±3

3

-

-

-

57±3

19±2

10

-

-

-

60±2

18±1

33

12±3

10±2

29±3

60±4

18±2

100

11±5

7±2

21±3

57±5

18±3

333

8±3

8±4

29±2

54±7

19±1

1000

13±1

4±3

27±3

40±0

17±2

3330 SP

11±3

1±1 **

24±4

24±5 **

17±3

5000 SP

-

-

-

18±2 **

16±1

 

With S9 *

Positive control

272±43

724±46

585±15

427±89

171±14

Solvent control

13±5

7±2

27±4

67±8

18±3

3

-

-

-

56±1

20±0

10

-

-

-

62±8

21±3

33

13±5

7±2

24±2

62±6

20±4

100

10±3

6±2

27±5

64±7

21±1

333

8±3

5±1

30±3

60±5

20±4

1000

13±3

7±4

28±3

49±5

17±2

3330

10±1

0±0

25±6

16±4 **

15±4

5000

-

-

-

19±6**

17±3

Solvent control: 0.1 ml dimethyl sulfoxide

*The S9-mix contained 5% (v/v) S9 fraction

**Bacterial background lawn slightly reduced

SP Slight Precipitate

- Not tested

Experiment 2: MUTAGENIC RESPONSE OF ZINN(II)-RICINOLEAT IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND IN THE ESCHERICHIA COLI REVERSE MUTATION ASSAY

Dose [µg/plate]

Mean number of revertant colonies/3 replicates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

 

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

 

Without S9

Positive control

410±6

246±62

494±41

709±116

171±41

Solvent control

12±2

4±1

15±2

67±11

15±3

33

8±4

5±2

16±3

92±18

8±4

100

12±2

3±1

18±3

83±3

7±2

333

13±5

3±1

19±2

83±6

7±1

1000

8±1

3±0

13±2

54±7

8±3

1670 SP

-

3±0

-

-

-

3330 SP

8±2

-

14±1

27±3 **

8±2

 

With S9 *

Positive control

258±19

245±14

541±19

728±66

349±20

Solvent control

13±3

3±1

22±4

66±6

14±3

33

12±3

5±1

20±2

76±7

8±3

100

15±3

5±1

24±3

74±1

7±2

333

14±2

5±3

19±3

64±9

7±3

1000

12±2

4±0

17±3

58±7

7±1

1670

-

5±1

-

-

-

3330

13±3

-

13±1

39±4 **

10±3

Solvent control: 0.1 ml dimethyl sulfoxide

*The S9-mix contained 10 % (v/v) S9 fraction

**Bacterial background lawn slightly reduced

SP Slight Precipitate

- Not tested

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that ZINN(II)-RICINOLEAT is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

ZINN(II)-RICINOLEAT was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coliWP2uvrA in two independent experiments.

In the dose range finding test, ZINN(II)-RICINOLEAT was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA 100 and WP2uvrA. ZINN(II)RICINOLEAT precipitated on the plates at dose levels of 3330 µg/plate and upwards. In tester strain WP2uvrA, the bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed. In tester strain TA 100, toxicity was observed at dose levels of 1000 µg/plate and upwards in the absence and presence of S9-mix.

In the first mutation experiment, ZINN(II)-RICINOLEAT was tested up to concentrations of 3330 µg/plate in the absence and presence of S9-mix in the strains TA1535, TA1537 and TA98. In the second mutation experiment, ZINN(II)-RICINOLEAT was tested up to concentrations of 3330 µg/plate in the absence and presence of S9-mix in the strains TA 1535, TA98, TA 100 and

WP2uvrA. ZINN(II)-RICINOLEAT was tested up to concentrations of 1670 µg/plate in the absence and presence of S9-mix in the strain TA1537.

ZINN(II)-RICINOLEAT did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA 1535, TA 1537, TA98 and TA 100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that ZINN(II)-RICINOLEAT is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.