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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:
27 November 2017 to 19 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Isobutylamine
EC Number:
201-145-4
EC Name:
Isobutylamine
Cas Number:
78-81-9
Molecular formula:
C4H11N
IUPAC Name:
isobutylamine
Test material form:
liquid
Specific details on test material used for the study:
A volume of 0.25 L of vapour was produced from 1 mL of the test item at 37°C. All concentrations cited in this report are expressed in terms of the CA3324A sample as received.

Method

Target gene:
The S. typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
E.coli WP2 pKM101 and WP2 uvrA pKM101
Details on mammalian cell type (if applicable):
The strains of S.typhimurium and E.coli were obtained from Moltox Inc. Batches of the strains were stored at -90 to -70°C as aliquots of nutrient broth cultures. DMSO was added to the cultures at 8% v/v as a cropreservative. Eachbatch of frozen strain was tested for amino acid requiremetn and , where applicable, for cell membrane permeability (rfa mutation), deficiency in DNA excision repair system (uvrA/uvrB mutation), and the pKM101 plasmid that confers resistantce to antibiotics. The responses of the strains to a series of reference mutagens were also assessed and were within the normal ranges determines in this laboratory.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment 1: Without and with S9 mix: 1.25%, 2.5%, 5%, 10%, 20%, 40%, 70% v/v test substance
Experiment 2: Without S9 mix: 0.08%, 0.16%, 0.31%, 0.63%, 1.25%, 2.5%, 5% v/v test substance
With S9 mix: 0.16%, 0.31%, 0.63%, 1.25%, 2.5%, 5% v/v test substance
Experiment 3: Without and with S9 mix: 0.08%, 0.16%, 0.31%, 0.63%, 1.25%, 2.5%, 5% v/v test substance
Experiment 4: Without and with S9 mix: 0.08%, 0.16%, 0.31%, 0.63%, 1.25%, 2.5%, 5% v/v test substance
Vehicle / solvent:
None
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
sterile air
Untreated negative controls:
yes
Remarks:
incubator controls
Remarks:
Concurrent incubatro controls treated with culture medium in the absence of the test item were placed in an incubator set to 34 to 39°C (i.e. cultures were not placed in the exposure vessels).
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9 mix
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-1-oxide
Remarks:
Without S9 mix
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
without S9 mix
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: The bacteria were exposed to measured volumes of the test item in sealed stainless steel vessels incubated at 34-37°C. Sterile air was introduced inside a class II microbiological safety cabinet to equilibrate to atmospheric pressure.
Four separate independent experiments were conducted in the absence and presence of S9 mix. S9mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10 hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and sodium phosphate buffer. The seeded plates were placed in stainless steel vessels. Plates containing S9 mix and buffer were placed in separate vessels. The vessels were sealed and partially evacuated. Appropriate volumes of the test substance were added to the vessels. The vessels were warmed to 34 to 39°C and the contents equilibrated to atmospheric pressure, where necessary, by admitting sterile atmospheric air. The plates were in cubated for ca 48 hours in the vessels at 34 to 39°C and then removed from the vessels under air extraction. The plates were incubated for a further period of ca 24 hours at 37°C to permit the growth of revertant colonies. Further sets of plates were prepared for the liquid positive control compounds. Aliquots of 0.1 mL of the positive control solutions were added to the plates together with the bacteria, buffer or S9 mix and agar overlay. These plates were incubated at 34 to 39°C for 48-72 hours (not in stainless steel vessels). After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter. Concentrations of the test substance up to 70% v/v per plate were tested. This is the maximum
practicable achievable concentration.

NUMBER OF REPLICATIONS: 3
Rationale for test conditions:
The strains were used to detect base changes and frameshift mutations as follows: Base change mutagens: S. typhimurium TA1535 and TA100, and E. coli WP2 uvrA (pKM101). Frameshift mutagens: S. typhimurium TA1537 and TA98. As the test item is known to be in vapour phase at the nominal temperature used within the test system (37°C) a modification of the test procedure was used to ensure consistent exposure.
Evaluation criteria:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory. The positive control compounds must induce an increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9/mL. A minimum of five analysable concentrations should be present with at least four showing no signs of toxic effects, evident as bacterial inhibition and/or a reduction in the number of revertants below the indication factor of 0.5. Mean number +SD of revertant colonies calculated for all groups. Fold-increases relative to vehicle controls calculated and means for treatment groups compared with vehicle control groups. It is considered to exhibit mutagenic activity if a reproducible increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, is seen. If exposure does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.

If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.
Statistics:
Not applicable

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additional info on results
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additinal info on results
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additional info on results
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additional infor on results
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see additional info on results
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the first experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test substance at 5% v/v per plate and above; except in TA98 in the absence of S9 mix and TA1535 in the absence and presence of S9 mix where toxicity was observed at 2.5% v/v per plate and above. Densely coloured plates were seen at 10% v/v and above.

In the second experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test substance at 5% v/v/plate. A greater than or equal to 2 fold-increase in revertant colonies was observed following exposure to the test substance at 0.16, 0.31, 0.63 and 1.25 % v/v per plate in the presence of S9 mix in strain TA1535 and 0.31 % v/v/plate in the absence of S9 mix in strain TA1535 and at 0.31 %v/v per plate in the absence of S9 mix in strain TA1537.

In the third experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test substance at 5% v/v per plate: except in strain TA1535 without S9 mix where toxicity was observed at 1.25 and 5% v/v per plate and in strain TA98 without S9 mix where toxicity occurred at 2.5% v/v per plate and above. No evidence of mutagenic activity was seen at any concentration of the test substance. There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In the fourth experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test substance at 5% v/v per plate: except in strain TA1537 in the absence of S9 mix where toxicity was observed at 2.5% v/v per plate and above. No evidence of mutagenic activity was seen at any concentration of the test substance. There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

As the results observed in the second experiment were not reproduced in the third and fourth independent experiments the overall result is that the test substance is considered not to be mutagenic.

Any other information on results incl. tables

Table 1: Experiment 1 without metabolic activation

Strain

Addition

Concentration plate

Mean revertants/plate

SD

Fold Increase (a)

TA98

Incubator Control

 

19.7

2.3

 

 

Vehicule

 

17.7

0.6

 

 

Test substance

1.25 % v/v

9.3

0.6

0.5

 

 

2.5 % v/v

6.3

0.6

0.4

 

 

5 % v/v

0.0

0.0

0.0

 

 

10 % v/v

0.0

0.0

0.0

 

 

20 % v/v

0.0

0.0

0.0

TA100

Incubator Control

 

152.7

31.1

 

 

Vehicule

 

134.3

1.5

 

 

Test substance

1.25 % v/v

101.0

14.1

0.8

 

 

2.5 % v/v

81.7

11.2

0.6

 

 

5 % v/v

0.0

0.0

0.0

 

 

10 % v/v

0.0

0.0

0.0

 

 

20 % v/v

0.0

0.0

0.0

TA1535

Incubator Control

 

16.7

8.1

 

 

Vehicule

 

17.7

2.5

 

 

Test substance

1.25 % v/v

10.3

4.6

0.6

 

 

2.5 % v/v

5.7

6.4

0.3

 

 

5 % v/v

0.3

0.6

0.0

 

 

10 % v/v

0.0

0.0

0.0

 

 

20 % v/v

0.0

0.0

0.0

TA1537

Incubator Control

 

10.3

3.8

 

 

Vehicule

 

7.3

2.5

 

 

Test substance

1.25 % v/v

12.0

5.2

1.6

 

 

2.5 % v/v

7.7

2.1

1.0

 

 

5 % v/v

0.0

0.0

0.0

 

 

10 % v/v

0.0

0.0

0.0

 

 

20 % v/v

0.0

0.0

0.0

WP2 uvrA (pKM101)

Incubator Control

 

108.3

7.0

 

 

Vehicule

 

114.0

24.9

 

 

Test substance

1.25 % v/v

122.0

137.7

1.1

 

 

2.5 % v/v

103.

6.5

0.9

 

 

5 % v/v

0.3

0.6

0.0

 

 

10 % v/v

0.0

0.0

0.0

 

 

20 % v/v

0.0

0.0

0.0

TA98

2NF

2 µg

265.3

38.4

15.0

TA100

NaN3

2 µg

623.3

46.1

4.6

TA1535

NaN3

2 µg

774.7

25.3

43.8

TA1537

AAC

50 µg

95.0

16.1

13.0

WP2 uvrA(pKM101)

NQO

2 µg

1471.7

92.7

12.9

 

2NF = 2-nitrofluoren; NaN3=sodium azide; AAC = aminoacridine; NQO = 4-Nitroquinoline-1-oxide, D = densely coloured plate; T= thinning of background lawn; (a) = relevant to vehicle. Plates at 40 and 70% v/v/plate were discarded after the 48 hour exposure to test substance.

 

Table 2: Experiment 1 with metabolic activation

Strain

Addition

Concentration plate

Mean revertants/plate

SD

Fold Increase (a)

TA98

Incubator Control

 

75.0

12.1

 

 

Vehicle

 

69.3

31.6

 

 

Test substance

1.25 % v/v

53.0

12.1

0.8

 

 

2.5 % v/v

64.3

12.9

0.9

 

 

5 % v/v

0.0

0.0

0.0

 

 

10 % v/v

0.0

0.0

0.0

 

 

20 % v/v

0.0

0.0

0.0

TA100

Incubator Control

 

193.3

11.7

 

 

Vehicle

 

196.3

3.8

 

 

Test substance

1.25 % v/v

297.0

6.2

1.5

 

 

2.5 % v/v

174.0

9.8

0.9

 

 

5 % v/v

0.0

0.0

0.0

 

 

10 % v/v

0.0

0.0

0.0

 

 

20 % v/v

0.0

0.0

0.0

TA1535

Incubator Control

 

15.3

6.7

 

 

Vehicle

 

30.0

3.5

 

 

Test substance

1.25 % v/v

24.3

2.3

0.8

 

 

2.5 % v/v

12.0

0.0

0.4

 

 

5 % v/v

0.0

0.0

0.0

 

 

10 % v/v

0.0

0.0

0.0

 

 

20 % v/v

0.0

0.0

0.0

TA1537

Incubator Control

 

14.0

4.6

 

 

Vehicle

 

17.7

7.6

 

 

Test substance

1.25 % v/v

24.0

2.0

1.4

 

 

2.5 % v/v

30.3

1.2

1.7

 

 

5 % v/v

0.0

0.0

0.0

 

 

10 % v/v

0.0

0.0

0.0

 

 

20 % v/v

0.0

0.0

0.0

WP2 uvrA (pKM101)

Incubator Control

 

231.0

22.3

 

 

Vehicle

 

202.3

7.4

 

 

Test substance

1.25 % v/v

219.7

5.5

1.7

 

 

2.5 % v/v

143.0

44.0

0.7

 

 

5 % v/v

0.0

0.0

0.0

 

 

10 % v/v

0.0

0.0

0.0

 

 

20 % v/v

0.0

0.0

0.0

TA98

B[a]P

5 µg

229.0

3.6

3.3

TA100

AAN

5 µg

2814.7

16.3

14.3

TA1535

AAN

5 µg

286.7

28.3

9.6

TA1537

B[a]P

5 µg

61.0

6.0

3.5

WP2 uvrA (pKM101)

AAN

10 µg

1122.3

63.4

5.5

 

B[a]P = benzo[a]pyrene; AAN : 2-aminoanthracene; D = densely coloured plate; T= thinning of background lawn; (a) = relevant to vehicle. Plates at 40 and 70% v/v/plate were discarded after the 48 hour exposure to test substance.

 

Table 3: Experiment 2 without metabolic activation

Strain

Addition

Concentration plate

Mean revertants/plate

SD

Fold Increase (a)

TA98

Incubator Control

 

21.3

4.5

 

 

Vehicle

 

19.0

6.6

 

 

Test substance

0.08 % v/v

18.7

1.2

1.0

 

 

0.16 % v/v

23.7

4.9

1.2

 

 

0.31 % v/v

23.7

9.3

1.8

 

 

0.63 % v/v

21.3

1.5

1.1

 

 

1.25 % v/v

18.7

3.1

1.0

 

 

2.5 % v/v

14.3

3.2

0.8

 

 

5 % v/v

0.7

0.6

0.0

TA100

Incubator Control

 

148.0

20.2

 

 

Vehicle

 

117.0

12.1

 

 

Test substance

0.08 % v/v

112.3

15.3

1.0

 

 

0.16 % v/v

118.0

22.3

1.0

 

 

0.31 % v/v

132.0

7.8

1.1

 

 

0.63 % v/v

126.0

13.5

1.1

 

 

1.25 % v/v

107.0

20.9

0.9

 

 

2.5 % v/v

102.7

21.1

0.9

 

 

5 % v/v

0.3

0.6

0.0

TA1535

Incubator Control

 

17.3

2.5

 

 

Vehicle

 

12.7

4.0

 

 

Test substance

0.08 % v/v

19.3

7.6

1.5

 

 

0.16 % v/v

17.3

3.2

1.4

 

 

0.31 % v/v

26.3

10.3

2.1

 

 

0.63 % v/v

24.3

17.2

1.9

 

 

1.25 % v/v

17.0

5.6

1.3

 

 

2.5 % v/v

6.7

5.8

0.5

 

 

5 % v/v

1.0

1.0

0.1

TA1537

Incubator Control

 

12.3

7.4

 

 

Vehicle

 

6.7

0.6

 

 

Test substance

0.08 % v/v

5.0

1.0

0.7

 

 

0.16 % v/v

8.3

7.5

1.3

 

 

0.31 % v/v

13.3

4.5

2.0

 

 

0.63 % v/v

13.0

9.2

1.9

 

 

1.25 % v/v

4.3

2.5

0.6

 

 

2.5 % v/v

4.3

3.8

0.6

 

 

5 % v/v

1.3

0.6

0.2

WP2 uvrA (pKM101)

Incubator Control

 

100.0

17.5

 

 

Vehicle

 

102.3

4.0

 

 

Test substance

0.08 % v/v

121.7

6.5

1.2

 

 

0.16 % v/v

103.3

2.5

1.0

 

 

0.31 % v/v

123.0

4.6

1.2

 

 

0.63 % v/v

117.0

7.2

1.1

 

 

1.25 % v/v

100.3

2.5

1.0

 

 

2.5 % v/v

85.3

25.7

0.8

 

 

5 % v/v

0.3

0.6

0.0

TA98

2NF

2 µg

254.0

79.4

13.4

TA100

NaN3

2 µg

654.3

49.0

5.6

TA1535

NaN3

2 µg

747.7

33.1

59.0

TA1537

AAC

50 µg

113.3

31.4

17.0

WP2 uvrA(pKM101)

NQO

2 µg

2020.0

271.6

19.7

 

2NF = 2-nitrofluoren; NaN3=sodium azide; AAC = aminoacridine; NQO = 4-Nitroquinoline-1-oxide; (a) rel to vehicle

Table 4: Experiment 2 with metabolic activation

Strain

Addition

Concentration plate

Mean revertants/plate

SD

Fold Increase (a)

TA98

Incubator Control

 

29.0

6.0

 

 

Vehicle

 

28.3

1.2

 

 

Test substance

0.16 % v/v

39.0

6.9

1.4

 

 

0.31 % v/v

41.3

16.4

1.5

 

 

0.63 % v/v

31.7

6.4

1.1

 

 

1.25 % v/v

22.7

15.0

0.8

 

 

2.5 % v/v

26.0

2.6

0.9

 

 

5 % v/v

0.3

0.6

0.0

TA100

Incubator Control

 

175.7

12.5

 

 

Vehicle

 

147.0

15.7

 

 

Test substance

0.16 % v/v

146.7

8.1

1.0

 

 

0.31 % v/v

165.3

10.2

1.1

 

 

0.63 % v/v

141.3

15.2

1.0

 

 

1.25 % v/v

135.3

4.7

0.9

 

 

2.5 % v/v

190.3

10.7

1.3

 

 

5 % v/v

1.3

2.3

0.0

TA1535

Incubator Control

 

12.0

2.6

 

 

Vehicle

 

11.3

3.2

 

 

Test substance

0.16 % v/v

23.7

2.5

2.1

 

 

0.31 % v/v

24.7

9.1

2.2

 

 

0.63 % v/v

24.0

7.2

2.1

 

 

1.25 % v/v

28.7

9.9

2.5

 

 

2.5 % v/v

12.7

0.6

1.1

 

 

5 % v/v

2.3

3.2

0.2

TA1537

Incubator Control

 

14.0

1.7

 

 

Vehicle

 

14.7

2.9

 

 

Test substance

0.16 % v/v

11.7

9.0

0.8

 

 

0.31 % v/v

15.3

9.2

1.0

 

 

0.63 % v/v

15.7

0.6

1.1

 

 

1.25 % v/v

7.7

1.2

0.5

 

 

2.5 % v/v

10.3

0.6

0.7

 

 

5 % v/v

3.3

2.9

0.2

WP2 uvrA (pKM101)

Incubator Control

 

148.0

13.1

 

 

Vehicle

 

148.0

14.8

 

 

Test substance

0.16 % v/v

138.0

6.0

0.9

 

 

0.31 % v/v

168.7

11.8

1.1

 

 

0.63 % v/v

144.7

18.1

1.0

 

 

1.25 % v/v

146.0

18.5

1.0

 

 

2.5 % v/v

151.7

17.0

1.0

 

 

5 % v/v

2.0

2.6

1.0

TA98

B[a]P

5 µg

185.7

24.7

6.6

TA100

AAN

5 µg

1888.0

139.1

12.8

TA1535

AAN

5 µg

270.7

18.5

23.9

TA1537

B[a]P

5 µg

68.7

3.1

4.7

WP2 uvrA(pKM101)

NQO

2 µg

1324.0

 

82.4

8.9

B[a]P = benzo[a]pyrene; AAN : 2-aminoanthracene;; T= thinning of background lawn24.7; (a) = relevant to vehicle.

Table 5: Experiment 3 without metabolic activation

Strain

Addition

Concentration plate

Mean revertants/plate

SD

Fold Increase (a)

TA98

Incubator Control

 

23.0

2.6

 

 

Vehicle

 

28.3

6.4

 

 

Test substance

0.08 % v/v

24.7

3.2

0.9

 

 

0.16 % v/v

22.7

8.5

0.8

 

 

0.31 % v/v

22.7

3.1

0.8

 

 

0.63 % v/v

16.7

6.1

0.6

 

 

1.25 % v/v

30.0

7.5

1.1

 

 

2.5 % v/v

9.7

4.0

0.3

 

 

5 % v/v

0.3

0.6

0.0

TA100

Incubator Control

 

190.7

11.9

 

 

Vehicle

 

149.7

7.5

 

 

Test substance

0.08 % v/v

126.7

29.3

0.8

 

 

0.16 % v/v

160.7

5.9

1.1

 

 

0.31 % v/v

145.0

2.0

1.0

 

 

0.63 % v/v

128.7

11.6

0.9

 

 

1.25 % v/v

131.7

11.0

0.9

 

 

2.5 % v/v

153.3

14.2

1.0

 

 

5 % v/v

0.0

0.0

0.0

TA1535

Incubator Control

 

18.0

3.0

 

 

Vehicle

 

32.0

3.0

 

 

Test substance

0.08 % v/v

24.7

9.1

0.8

 

 

0.16 % v/v

22.0

11.3

0.7

 

 

0.31 % v/v

21.0

8.7

0.7

 

 

0.63 % v/v

20.7

6.4

0.6

 

 

1.25 % v/v

14.0

3.6

0.4

 

 

2.5 % v/v

18.3

1.5

0.6

 

 

5 % v/v

0.0

0.0

0.0

TA1537

Incubator Control

 

12.3

5.0

 

 

Vehicle

 

6.6

0.6

 

 

Test substance

0.08 % v/v

7.0

0.0

1.1

 

 

0.16 % v/v

8.0

1.7

1.3

 

 

0.31 % v/v

12.0

1.7

1.9

 

 

0.63 % v/v

9.0

5.3

1.4

 

 

1.25 % v/v

8.3

1.2

1.3

 

 

2.5 % v/v

7.7

2.9

1.2

 

 

5 % v/v

0.0

0.0

0.0

WP2 uvrA (pKM101)

Incubator Control

 

184.3

7.8

 

 

Vehicle

 

148.7

9.5

 

 

Test substance

0.08 % v/v

159.7

21.2

1.1

 

 

0.16 % v/v

180.7

19.6

1.2

 

 

0.31 % v/v

186.3

15.5

1.3

 

 

0.63 % v/v

165.0

10.0

1.1

 

 

1.25 % v/v

190.0

15.4

1.3

 

 

2.5 % v/v

185.0

14.7

1.2

 

 

5 % v/v

2.7

2.1

0.0

TA98

2NF

2 µg

275.0

17.1

9.7

TA100

NaN3

2 µg

602.7

61.0

4.0

TA1535

NaN3

2 µg

819.3

24.4

25.6

TA1537

AAC

50 µg

156.7

51.4

24.7

WP2 uvrA(pKM101)

NQO

2 µg

2111.3

297.3

14.2

 

2NF = 2-nitrofluoren; NaN3=sodium azide; AAC = aminoacridine; NQO = 4-Nitroquinoline-1-oxide; (a) rel to vehicle

Table 6: Experiment 3 with metabolic activation

Strain

Addition

Concentration plate

Mean revertants/plate

SD

Fold Increase (a)

TA98

Incubator Control

 

34.0

3.0

 

 

Vehicle

 

25.7

10.0

 

 

Test substance

0.08 % v/v

28.3

4.0

1.1

 

 

0.16 % v/v

32.7

13.3

1.3

 

 

0.31 % v/v

22.0

6.2

0.9

 

 

0.63 % v/v

29.7

15.2

1.2

 

 

1.25 % v/v

26.0

2.6

1.0

 

 

2.5 % v/v

19.0

3.5

0.7

 

 

5 % v/v

0.7

1.2

0.0

TA100

Incubator Control

 

161.3

17.4

 

 

Vehicle

 

126.3

20.2

 

 

Test substance

0.08 % v/v

129.7

5.5

1.0

 

 

0.16 % v/v

113.3

39.4

0.9

 

 

0.31 % v/v

143.3

15.9

1.1

 

 

0.63 % v/v

149.3

5.1

1.2

 

 

1.25 % v/v

147.7

7.5

1.2

 

 

2.5 % v/v

143.3

20.6

1.1

 

 

5 % v/v

0.7

0.6

0.0

TA1535

Incubator Control

 

13.0

1.7

 

 

Vehicle

 

26.0

6.1

 

 

Test substance

0.08 % v/v

22.7

1.2

0.9

 

 

0.16 % v/v

15.3

2.9

0.6

 

 

0.31 % v/v

13.7

4.0

0.5

 

 

0.63 % v/v

23.3

4.9

0.9

 

 

1.25 % v/v

26.3

2.5

1.0

 

 

2.5 % v/v

19.3

6.4

0.7

 

 

5 % v/v

0.3

0.6

0.0

TA1537

Incubator Control

 

12.3

4.6

 

 

Vehicle

 

13.3

3.8

 

 

Test substance

0.08 % v/v

17.7

9.3

1.3

 

 

0.16 % v/v

15.3

11.0

1.1

 

 

0.31 % v/v

19.7

1.5

1.5

 

 

0.63 % v/v

12.3

5.5

0.9

 

 

1.25 % v/v

10.0

3.6

0.7

 

 

2.5 % v/v

8.3

2.9

0.6

 

 

5 % v/v

0.0

0.0

0.0

WP2 uvrA (pKM101)

Incubator Control

 

120.7

5.9

 

 

Test substance

0.08 % v/v

131.0

27.9

1.0

 

 

0.16 % v/v

124.7

41.2

0.9

 

 

0.31 % v/v

128.3

11.6

1.0

 

 

0.63 % v/v

141.3

19.1

1.1

 

 

1.25 % v/v

132.3

19.6

1.0

 

 

2.5 % v/v

108.3

7.4

0.8

 

 

5 % v/v

2.3

3.2

0.0

TA98

B[a]P

5 µg

193.3

5.5

7.5

TA100

AAN

5 µg

1629.0

243.6

12.9

TA1535

AAN

5 µg

311.3

20.6

12.0

TA1537

B[a]P

5 µg

75.0

12.8

5.6

WP2 uvrA(pKM101)

NQO

2 µg

760.3

28.4

5.8

B[a]P = benzo[a]pyrene; AAN : 2-aminoanthracene;; T= thinning of background lawn24.7; (a) = relevant to vehicle.

Table 7: Experiment 4 without metabolic activation

Strain

Addition

Concentration plate

Mean revertants/plate

SD

Fold Increase (a)

TA98

Incubator Control

 

19.7

3.5

 

 

Vehicle

 

22.0

2.6

 

 

Test substance

0.08 % v/v

118.7

3.8

0.8

 

 

0.16 % v/v

21.3

3.1

1.0

 

 

0.31 % v/v

16.7

1.2

0.8

 

 

0.63 % v/v

25.0

5.0

1.1

 

 

1.25 % v/v

19.0

4.4

0.9

 

 

2.5 % v/v

13.0

3.5

0.6

 

 

5 % v/v

0.0

0.0

0.0

TA100

Incubator Control

 

132.0

4.0

 

 

Vehicle

 

138.7

3.2

 

 

Test substance

0.08 % v/v

140.7

11.0

1.0

 

 

0.16 % v/v

136.0

7.2

1.0

 

 

0.31 % v/v

135.7

5.7

1.0

 

 

0.63 % v/v

141.7

2.1

0.9

 

 

1.25 % v/v

120.3

4.2

1.0

 

 

2.5 % v/v

132.3

4.5

0.0

 

 

5 % v/v

0.0

0.0

 

TA1535

Incubator Control

 

14.7

1.5

 

 

Vehicle

 

16.3

5.9

0.9

 

Test substance

0.08 % v/v

15.3

1.5

1.1

 

 

0.16 % v/v

18.0

1.7

1.1

 

 

0.31 % v/v

17.3

1.5

1.1

 

 

0.63 % v/v

18.3

4.0

1.1

 

 

1.25 % v/v

12.3

4.0

0.8

 

 

2.5 % v/v

15.0

2.6

0.9

 

 

5 % v/v

0.0

0.0

0.0

TA1537

Incubator Control

 

17.0

2.6

 

 

Vehicle

 

16.3

4.7

 

 

Test substance

0.08 % v/v

22.0

1.0

1.3

 

 

0.16 % v/v

20.7

505

1.3

 

 

0.31 % v/v

26.0

3.5

1.6

 

 

0.63 % v/v

21.3

4.7

1.3

 

 

1.25 % v/v

16.0

4.4

1.0

 

 

2.5 % v/v

3.0

1.7

0.2

 

 

5 % v/v

0.0

0.0

0.0

WP2 uvrA (pKM101)

Incubator Control

 

122.0

3.0

 

 

Vehicle

 

123.3

2.1

 

 

Test substance

0.08 % v/v

109.3

2.5

0.9

 

 

0.16 % v/v

116.0

4.6

0.9

 

 

0.31 % v/v

118.3

2.1

1.0

 

 

0.63 % v/v

115.3

1.5

0.9

 

 

1.25 % v/v

115.0

3.0

0.9

 

 

2.5 % v/v

99.7

3.1

0.8

 

 

5 % v/v

0.7

1.2

0.0

TA98

2NF

2 µg

283.3

48.8

12.9

TA100

NaN3

2 µg

544.7

17.2

3.9

TA1535

NaN3

2 µg

812.7

40.8

49.8

TA1537

AAC

50 µg

193.3

62.0

11.8

WP2 uvrA(pKM101)

NQO

2 µg

1880.3

63.4

15.2

 

2NF = 2-nitrofluoren; NaN3=sodium azide; AAC = aminoacridine; NQO = 4-Nitroquinoline-1-oxide; (a) rel to vehicle

Table 8: Experiment 4 with metabolic activation

Strain

Addition

Concentration plate

Mean revertants/plate

SD

Fold Increase (a)

TA98

Incubator Control

 

37.7

3.5

 

 

Vehicle

 

40.3

12.1

 

 

Test substance

0.08 % v/v

37.0

6.0

0.9

 

 

0.16 % v/v

36.0

5.2

0.9

 

 

0.31 % v/v

35.7

8.1

0.9

 

 

0.63 % v/v

40.7

1.2

1.0

 

 

1.25 % v/v

29.0

14.7

0.7

 

 

2.5 % v/v

21.3

4.6

0.5

 

 

5 % v/v

0.0

0.0

0.0

TA100

Incubator Control

 

175.7

8.1

 

 

Vehicle

 

157.3

22.8

 

 

Test substance

0.08 % v/v

166.0

9.5

1.1

 

 

0.16 % v/v

174.0

8.5

1.1

 

 

0.31 % v/v

163.3

117.6

1.0

 

 

0.63 % v/v

170.3

9.1

1.1

 

 

1.25 % v/v

153.0

24.8

1.0

 

 

2.5 % v/v

150.7

30.4

1.0

 

 

5 % v/v

3.3

2.1

0.0

TA1535

Incubator Control

 

15.3

6.0

 

 

Vehicle

 

11.0

3.5

 

 

Test substance

0.08 % v/v

12.3

0.6

1.1

 

 

0.16 % v/v

9.0

0.0

0.8

 

 

0.31 % v/v

13.3

2.9

1.2

 

 

0.63 % v/v

14.7

1.5

1.3

 

 

1.25 % v/v

11.3

1.5

1.0

 

 

2.5 % v/v

11.7

2.9

1.1

 

 

5 % v/v

0.3

0.6

0.0

TA1537

Incubator Control

 

16.0

5.3

 

 

Vehicle

 

13.0

3.0

 

 

Test substance

0.08 % v/v

14.3

2.3

1.1

 

 

0.16 % v/v

15.3

4.9

1.2

 

 

0.31 % v/v

10.7

1.2

0.8

 

 

0.63 % v/v

10.0

2.6

0.8

 

 

1.25 % v/v

7.3

1.5

0.6

 

 

2.5 % v/v

11.0

6.6

0.8

 

 

5 % v/v

0.3

0.6

0.0

WP2 uvrA (pKM101)

Incubator Control

 

194.0

18.5

 

 

Vehicle

 

171.0

13.0

 

 

Test substance

0.08 % v/v

210.7

22.6

1.2

 

 

0.16 % v/v

191.7

204.4

1.1

 

 

0.31 % v/v

193.0

5.3

1.1

 

 

0.63 % v/v

195.0

18.2

1.1

 

 

1.25 % v/v

189.0

22.6

1.1

 

 

2.5 % v/v

178.3

3.5

1.0

 

 

5 % v/v

0.0

0.0

0.0

TA98

B[a]P

5 µg

187.7

8.5

4.7

TA100

AAN

5 µg

1729.0

264.2

11.0

TA1535

AAN

5 µg

345.0

5.2

31.4

TA1537

B[a]P

5 µg

70.7

4.6

5.4

WP2 uvrA(pKM101)

NQO

2 µg

730.0

23.6

4.3

B[a]P = benzo[a]pyrene; AAN : 2-aminoanthracene;; T= thinning of background lawn24.7; (a) = relevant to vehicle.

 

 

Applicant's summary and conclusion

Conclusions:
The test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed. Hence, the test item is concluded to be negative, i.e. non-mutagenic, in this assay.
Executive summary:

Histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Eschrichi coli, stain WPU uvrA (pKM101) were wxposed to the test substance vapour diluted in sterile air in a vapour phase system. Sterile air was used as a vehicle control.

Four independent mutation experiments were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The experiments were modified plate incorporation assays using a vapour phase system.

Concentrations of the test item up to 70% v/v/plate were tested. This is the maximum practicable achievable concentration. A series of lower concentration were also used.

In the first experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test item at 5% v/v/plate and above; except in TA98 in the absence of S9 mix and TA1535 in the absence and presence of S9 mix where toxicity was observed at 2.5% v/v/plate and above. Densely coloured plates were seen at 10% v/v and above.

In the second experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test item at 5% v/v/plate. A greater than or equal to 2 fold-increase in revertant colonies was observed following exposure to the test item at 0.16, 0.31, 0.63 and 1.25 % v/v/plate in the presence of S9 mix in strain TA1535 and 0.31 % v/v/plate in the absence of S9 mix in strain TA1535 and at 0.31 %v/v/plate in the absence of S9 mix in strain TA1537.

In the third experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to CA3324A at 5% v/v/plate: except in strain TA1535 without S9 mix where toxicity was observed at 1.25 and 5% v/v/plate and in strain TA98 without S9 mix where toxicity occurred at 2.5% v/v/plate and above. No evidence of mutagenic activity was seen at any concentration of CA3324A. There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In the fourth experiment, toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or a reduction in revertant colony numbers (below an induction factor of 0.5)) was seen in all strains following exposure to the test item at 5% v/v/plate: except in strain TA1537 in the absence of S9 mix where toxicity was observed at 2.5% v/v/plate and above. No evidence of mutagenic activity was seen at any concentration of the test item. There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

As the results observed in the second experiment were not reproduced in the third and fourth independent experiments the overall result is that the test item is considered not to be mutagenic.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in revertant colonies, consistent with the historial positive control range for the laboratory, verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle and incubator controls were within the current historical control range for the laboratory.

It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed. Hence, the test item is concluded to be negative, i.e. non-mutagenic, in this assay.