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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 December 1980 to 08 December 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The bacterial strains tested differ slightly from the guideline.
GLP compliance:
no
Remarks:
Study predates GLP
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): C192 SN 2188-2; Aldehyde C9 Nonylic
- Appearance: Clear colourless liquid

Method

Species / strain
Species / strain / cell type:
other: TA-1535, TA-1537, TA-1538, TA-98 and TA-100
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal enzyme preparations from Aroclor induced rats
Test concentrations with justification for top dose:
Initial assay: 0, 0.01, 0.1, 1.0, 5.0, 10.0, 25.0 and 50.0 µL per plate
Definitive assay: 0, 0.0001, 0.0005, 0.001, 0.005 and 0.01 µL per plate
Vehicle / solvent:
- Solvent used: DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA-1537: without metabolic activation; 50 µg/plate
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA-1535 and TA-100: without metabolic activation; 10 µg/plate
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA-1538 and TA-98: without metabolic activation; 10 µg/plate
Positive controls:
yes
Positive control substance:
other: 2-anthramine, all strains: with metabolic activation; 2.5 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

MEDIA
The bacterial strains were cultured in Oxoid Media #2 (nutrient broth). The selective medium was Vogel Bonner Medium E with 2% glucose. The overlay agar consisted of 0.6% purified agar with 0.5 mM histidine, 0.05 mM biotin and 0.1 M NaCl.

METABOLIC ACTIVATION SYSTEM
Preparation of S9 homogenate:
- A 9000xg supernatant prepared from Sprague-Dawley adult male rat liver induced by Aroclor 1254 was used in this assay.
Preparation of S9-Mix:
- S9-mix contained, per mL, 4 µmoles NADP, 5 µmoles D-glucose-6-phosphate, 100 µmoles sodium phosphate buffer pH 7.4; 8 µmoles MgCl₂, 33 µmoles KCl and 100 µL organ homogenate from rat liver (S9 fraction).

TEST PROCEDURE
a). Non-activation assay:
The following was added to a sterile 13x100 mm test tube placed in a 43 °C water bath:
- 2 mL of 0.6% agar containing 0.05 mM histidine and 0.05 mM biotin
- 0.05 mL of a solution of the test chemical to give the appropriate dose
- 0.1 - 0.2 mL of the indicator organism
- 0.5 mL of 0.2 M phosphate buffer, pH 7.4
The mixture was swirled gently and then poured onto minimal agar plates. After the top agar had set, the plates were incubated at 37 °C for approximately 2 days. The number of his+ revertant colonies growing on the plates was counted and recorded.
b). Activation assay:
The activation assay was run concurrently with the non-activation assay. The only difference was the addition of 0.5 mL of S9 mix to the tubes in the place of 0.5 mL of phosphate buffer.
Evaluation criteria:
1). Strains TA-1535, TA-1537 and TA-1538:
If the solvent control value was within the normal range, a test material producing a positive response equal to 3 times the solvent control is considered mutagenic.

2). Strains TA-98 and TA-100:
If the solvent control value was within the normal range, a test material producing a positive response equal to twice the solvent control is considered mutagenic.

If a test material produced a response in a single test which couldn't be reproduced in additional runs, the initial positive test data loses significance.

Results and discussion

Test results
Species / strain:
other: TA-1535, TA-1537, TA-1538, TA-98 and TA-100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material was toxic to the strains TA-1537 and TA-1538 at doses of 0.01 µL and above and to TA-98 at doses of 1.0 µL and above.

In the initial assays, indicator strains TA-1535 and TA-100 were not used because these 2 strains were contaminated with other types of bacteria. The test was subsequently repeated with all the indicator strains in the nonactivation and activation assays and the dose range employed was from 0.0001 µL to 0.01 µL per plate based on the toxicity observed with the strains TA-537, TA-1538 and TA-98. The test material was toxic to all strains except TA-98 and TA-100 at 0.01 µL dose in the repeat test.

The results of the tests conducted on the compound in both the presence and absence of a metabolic system were negative.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Table 1: Initial assay

Test

Concentration

(µL per plate)

Revertants per plate

TA-1537

TA-1538

TA-98

Non-activation

Solvent control

-

5

14

25

Solvent control

-

7

16

31

Positive control *

-

229

1261

828

Positive control *

-

234

1336

924

Test compound

0.01

0

0

27

0.1

0

0

28

1.0

1

0

0

5.0

0

0

0

10.0

0

0

0

25.0

0

0

0

50.0

0

0

0

Activation

Solvent control

-

9

25

32

Solvent control

-

9

30

51

Positive control **

-

141

1028

1143

Positive control **

-

203

1625

1193

Test compound

0.01

3

18

24

0.1

5

14

27

1.0

1

0

2

5.0

0

0

0

10.0

0

0

0

25.0

0

0

0

50.0

0

0

0

*:

TA-1537: 9-aminoacridine (50 µg/plate)

TA-1538: 2-nitrofluorene (10 µg/plate)

TA-98: 2-nitrofluorene (10 µg/plate)

 

**:

TA-1537: 2-anthramine (2.5 µg/plate)

TA-1538: 2-anthramine (2.5 µg/plate)

TA-98: 2-anthramine (2.5 µg/plate)

 

Solvent: 50 µL/plate

 

 

Table 2: Definitive assay

Test

Concentration

(µL per plate)

Revertants per plate

TA-1535

TA-1537

TA-1538

TA-98

TA-100

Non-activation

Solvent control

-

27

10

18

34

117

Solvent control

-

29

14

10

46

120

Positive control *

-

1468

463

1445

704

950

Positive control *

-

1497

520

1396

717

1075

Test compound

0.0001

24

13

12

29

113

0.0005

22

9

13

22

123

0.001

27

11

16

27

124

0.005

21

9

18

31

120

0.01

0

0

0

21

86

Activation

Solvent control

-

24

11

13

32

112

Solvent control

-

26

12

25

34

115

Positive control **

-

233

295

1681

629

1852

Positive control **

-

204

285

1135

1216

1711

Test compound

0.0001

29

12

25

30

116

0.0005

24

13

24

36

124

0.001

24

11

18

27

123

0.005

29

12

20

20

128

0.01

12

5

11

21

102

*:

TA-1535: sodium azide (10 µg/plate)

TA-1537: 9-aminoacridine (50 µg/plate)

TA-1538: 2-nitrofluorene (10 µg/plate)

TA-98: 2-nitrofluorene (10 µg/plate)

TA-100: sodium azide (10 µg/plate)

 

**:

TA-1535: 2-anthramine (2.5 µg/plate)

TA-1537: 2-anthramine (2.5 µg/plate)

TA-1538: 2-anthramine (2.5 µg/plate)

TA-98: 2-anthramine (2.5 µg/plate)

TA-100: 2-anthramine (2.5 µg/plate)

 

Solvent: 50 µL/plate

 

Applicant's summary and conclusion

Conclusions:
The test substance was assessed for genotoxicity according to OECD 471 with and without metabolic activation. The test material did not exhibit genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under the test conditions.