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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:
2003-09-03 to 2003-11-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Nonane-1,9-diol
EC Number:
223-517-5
EC Name:
Nonane-1,9-diol
Cas Number:
3937-56-2
Molecular formula:
C9H20O2
IUPAC Name:
nonane-1,9-diol
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Chemical name: 1,9-Nonanediol
- CAS no.: 3937-56-2
- Source and lot/batch No.of test material: : BASF / 42638
- Expiration date of the lot/batch: March 2003
- Molecular weight: 160.254 g/mol
- Purity: 98.8%

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 (prepared in house)
Test concentrations with justification for top dose:
1st Experiment (plate incorporation) and Experiment 2 (pre-incubation): 0, 20, 100, 500, 2500, 5000 ug/plate (the maximum recommended concentration in accordance with current regulatory guidelines for in vitro bacterial genotoxicity assays)
Due to toxicity observed at 5000 ug/plate in Experiment 2, a further pre-incubation test was undertaken (Experiment 3): 0, 250, 500, 750, 1000 and 1500 ug/plate (maximum concentration limited by toxicity)
Vehicle / solvent:
Dimethyl sulphoxide (DMSO)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: MNNG, NOPD, AAC, 4-NQO
Details on test system and experimental conditions:
Test tubes containing 2 mL of soft agar (overlay agar) kept at 45°C in a water bath had the following added: 0.1 mL test article formulation (or vehicle) ; 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (or PBS in the absence of metabolic activation).
After mixing Salmonella typhimurium containing tubes were poured onto Vogel-Bonner agar plates (minimal glucose agar plates). E.coli containing tubes were poured onto minimal agar plates.
Agar plates were incubated at 37°C for 48-72 h in the dark for the bacterial colonies (his+ or typ+ revertants) counted.
Pre-incubation:
0.1 mL of test article formulation (or vehicle), 0.1 mL bacterial suspension and 0.5 mL S9 mix (or PBS in the absence of metabolic activation) were incubated at 37°C for the duration of ~20 minutes. Subsequently, 2 mL of soft agar was added and samples were poured onto the agar plates.
In both cases, agar plates were incubated at 37°C for 48-72 h in the dark for the bacterial colonies (his+ or typ+ revertants) counted.

The background lawns of the plates were examined for signs of toxicity. Other toxicity indicators that may have been noted included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.
Evaluation criteria:
The test chemical was considered positive in this assay if the following criteria were met:
- dose-related and reproducible increase in the number of revertant colonies (i.e. doubling of the spontaneous mutation rate in at least one tester strain either –S9 or +S9)

A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Statistics:
Statistics not warranted

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

A. Mutation assay:

Experiment 1 (plate incorporation) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 1,9-Nonanediol at 20, 100, 500, 2500, 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity (evidenced by reduction in revertant numbers) was observed on the mutation plates treated at 5000 µg/mL with TA1537 and TA98 in the presence of S9. 1,9-Nonandiol was not mutagenic following a plate incorporation methodology

Experiment 2 (pre-incubation) treatments of all the tester strains were performed in the absence and presence of S-9 using the same dose concentrations as Experiment 1. Toxicity was evident at concentrations of 2500 ug/plate and greater. Consequently with only 4 scorable dose levels, a further pre-incubation experiment was undertaken to better define mutagenicity in the absence of overt toxicity.

Experiment 3 (pre-incubation) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 1,9-Nonanediol at 250, 500, 750, 1000, 1500 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity (evidenced by both a reduction in revertant numbers and background lawn) was observed on the mutation plates treated at 1500 µg/mL with TA98 and WP2uvrA in the absence of S9. 1,9-Nonanediol was not mutagenic following a pre-incubation methodology

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

The positive controls induced an acceptable increase in revertant colony numbers,thereby demonstrating the sensitivity and specificity of the test system.

Following1,9-Nonandioltreatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were equal to or greater than 2-fold above the concurrent vehicle control. This study was therefore considered to have provided no evidence of any mutagenic activity in this assay system (refer to Table 7.6.1/01-1, -2, -3).

Table 7.6.1/01-1:
Bacterial (reverse) gene mutation plate incorporation data – Experiment 1

Conc
(µg/plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

25

39

118

109

18

17

9

11

32

36

20

26

36

121

122

15

15

8

7

30

30

100

26

35

132

141

15

14

10

8

29

32

500

28

33

134

123

14

14

8

9

26

29

2500

23

36

127

128

10

10

6

6

24

29

5000

20

19

93

84

11

11

6

6

19

29

+ve

689

545

1117

986

907

128

459

113

614

253

+ve controls:

-S9 (absence of metabolic activation):

TA98: 4-nitro-o-phenylendiamine (NOPD)

TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

TA1537: 9-aminoacridine (AAC)

WP2uvrA: 4-nitroquinoline-N-oxide (4-NQO)

 

+S9 (presence of metabolic activation):

All strains: 2-aminoanthracene

Table 7.6.1/01-2:
Bacterial (reverse) gene mutation pre-incubation data – Experiment 2

Conc
(µg/plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

27

31

107

107

17

16

10

9

30

30

20

22

32

106

102

19

16

10

7

31

28

100

21

29

108

98

15

13

8

7

34

25

500

22

24

100

99

15

13

5

6

30

28

2500

10B

16B

43B

93B

2B

11B

1B

6B

26B

22

5000

0B

12B

0B

56B

0B

5B

0B

2B

0B

18B

+ve

802

603

805

846

719

91

379

118

634

242

B: reduced background lawn

+ve controls:

-S9 (absence of metabolic activation):

TA98: 4-nitro-o-phenylendiamine (NOPD)

TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

TA1537: 9-aminoacridine (AAC)

WP2uvrA: 4-nitroquinoline-N-oxide (4-NQO)

 

 

+S9 (presence of metabolic activation):

All strains: 2-aminoanthracene

Table 7.6.1/01-3:
Bacterial (reverse) gene mutation pre-incubation data – Experiment 3

Conc
(µg/plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

26

32

108

116

19

16

8

9

30

37

250

22

27

102

110

18

15

8

8

26

37

500

30

34

112

104

16

13

6

7

20

30

750

21

31

102

102

14

14

7

7

26

29

1000

26

25

101

105

16

14

7

7

23

27

1500

14

19

101

75

14

10

5

5

12

22

+ve

620

792

717

842

717

106

415

126

628

232

+ve controls:

-S9 (absence of metabolic activation):

TA98: 4-nitro-o-phenylendiamine (NOPD)

TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

TA1537: 9-aminoacridine (AAC)

WP2uvrA: 4-nitroquinoline-N-oxide (4-NQO)

 

+S9 (presence of metabolic activation):

All strains: 2-aminoanthracene

B. Deficiencies:

A single positive control, 2-aminoanthracene was used to evaluate the sensitivity of the assay in both the presence of S9-mix. This positive control may be activated by enzymes other than the microsomal cytochrome P450 family. Therefore it could be concluded that the S9 activity has not been demonstrated. However, S9 was prepared in house and the efficacy of the S9 mix was characterised with benzo[a]pyrene. Overall it is concluded that the S9 activity was adequately demonstrated.

Applicant's summary and conclusion

Conclusions:
It was concluded that 1,9-Nonanediol did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2uvrA) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/mL (the maximum recommended concentration according to current regulatory guidelines, and in several cases a toxic treatment concentration), in the absence and presence of a rat liver metabolic activation system (S9) using both plate incorporation and pre-incubation methodologies.
Executive summary:

In a reverse gene mutation assay in bacteria, S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA were exposed to1,9-Nonanediol formulated in dimethyl sulphoxide.Both plate incorporation and pre-incubation methodologies were used.

Experiment 1 (plate incorporation) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 1,9-Nonandiol at 20, 100, 500, 2500, 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity (evidenced by reduction in revertant numbers) was observed on the mutation plates treated at 5000 µg/mL with TA1537 and TA98 in the presence of S9. 1,9-Nonandiol was not mutagenic following a plate incorporation methodology

Experiment 2 (pre-incubation) treatments of all the tester strains were performed in the absence and presence of S-9 using the same dose concentrations as Experiment 1. Toxicity was evident at concentrations of 2500 ug/plate and greater. Consequently with only 4 scorable dose levels, a further pre-incubation experiment was undertaken to better define mutagenicity in the absence of overt toxicity.

Experiment 3 (pre-incubation) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 1,9-Nonandiol at 250, 500, 750, 1000, 1500 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity (evidenced by both a reduction in revertant numbers and background lawn) was observed on the mutation plates treated at 1500 µg/mL with TA98 and WP2uvrA in the absence of S9. 1,9-Nonandiol was not mutagenic following a pre-incubation methodology

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

The positive controls induced an acceptable increase in revertant colony numbers, thereby demonstrating the sensitivity and specificity of the test system.

Following 1,9-Nonanediol treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were equal to or greater than 2-fold above the concurrent vehicle control. This study was therefore considered to have provided no evidence of any mutagenic activity in this assay system.

It was concluded that 1,9-Nonanediol did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2uvrA) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/mL (the maximum recommended concentration according to current regulatory guidelines, and in several cases a toxic treatment concentration), in the absence and presence of a rat liver metabolic activation system (S9) using both plate incorporation and pre-incubation methodologies.

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