Registration Dossier

Diss Factsheets

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP condition

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996

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:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Salicylaldehyde
EC Number:
201-961-0
EC Name:
Salicylaldehyde
Cas Number:
90-02-8
Molecular formula:
C7H6O2
IUPAC Name:
salicylaldehyde
Details on test material:
- Name of test material(as cited in study report): 2-Hydroxybenzaldehyde - Physical state: pale yellowish liquid - Analytical purity: over 95 %( no information on the impurities): unknown - Supplier: SEIMI Chemical Co. Ltd. - Lot/batch No.: 082316- Stability: the stability of 2-hydroxybenzaldehyde in DMSO at concentrations of 156 ug/mL and 40 mg/mL were confirmed after 4-hour storage in a light -shielding condition at room temperature; the concentrations of 2-hydroxyaldehyde were reported 98.7-100 % at 156 ug/mL and 96.8 -99.8% at 40.0 mg/mL, compared with the concentrations at the time of preparation (0 hour)

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
SD Rat (7 week-old, male) liver induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
S9 mix: 0, 31.3, 62.5, 125, 250, 500 and 1000 μg/plate (TA1535, TA98 and TA1537) 0, 15.6, 31.3, 62.5, 125, 250 and 500 μg/plate (TA100) 0, 125, 250, 500, 1000, 2000 and 4000 μg/plate (WP2 uvrA) +S9 mix: 0, 62.5, 125, 250, 500, 1000 and 2000 μg/plate (TA1535, TA98 and TA1537) 0, 15.6, 31.3, 62.5, 125, 250 and 500 μg/plate (TA100) 0, 125, 250, 500, 1000, 2000 and 4000 μg/plate (WP2 uvrA)
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO - Justification for choice of solvent/vehicle: The test substance was highly soluble in DMSO.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: +S9 mix: 2-Aminoanthracene (all strains)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: S9 mix: AF-2 (TA100, TA98 and WP2 uvrA), Sodium azide (TA1535) and 9-Aminoacridine (TA1537)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) DURATION - Exposure duration: 48 hrs NUMBER OF PLATES: 3 NUMBER OF REPLICATIONS: 2 DETERMINATION OF CYTOTOXICITY - Method: other: growth inhibition 
Evaluation criteria:
In any strain(s) tested with or without S9 mix, when the mean number of revertant colonies per plate increased twice more than that of the negative control and when the increase was shown to be dose-related and reproducible, the chemical was judged to be mutagenic. 

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA100, TA1535, TA98, TA1537 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Concentration range: 50 - 5000 μg/plate (common ratio: 3) Cytotoxic conc.: -S9 mix: >=1500 μg/plate (TA1535, TA98, TA1537); >=500 μg/plate (TA100); 5000 μg/plate (WP2 uvrA) +S9 mix: >=1500 μg/plate (TA1535, TA98, TA1537 and WP2uvrA); >=500 μg/plate (TA100)ADDITIONAL INFORMATION ON CYTOTOXICITY: [1st trial] -S9 mix: 4000 μg/plate (WP2 uvrA); 1000 μg/plate (TA1535, TA98, TA1537); 500 μg/plate (TA100) +S9 mix: 4000 μg/plate (WP2 uvrA); 2000 μg/plate (TA1535, TA98, TA1537); 500 μg/plate (TA100) [2nd trial] -S9 mix: 4000 μg/plate (WP2 uvrA); 1000 μg/plate (TA1535, TA98, TA1537); 500 μg/plate (TA100) +S9 mix: 4000 μg/plate (WP2 uvrA); 2000 μg/plate (TA98, TA1537); 1000 μg/plate (TA1535); 500 μg/plate (TA100) 
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

[1st trial] See Table 1

-S9 mix: Negative (all test strains)

+S9 mix: Negative (all test strains)

[2nd trial] See Table 2

-S9 mix: Negative (all test strains)

+S9 mix: Negative (all test strains)

Table 1 Mutagenicity of 2-hydroxybenzaldehyde** in reverse mutation test (I) on bacteria

With(+) orwithout(-)S9 mix

Test substancedose(μg/plate)

Number of revertants (Number of colonies/plate, Mean ±S.D.)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

S9 mix(-)

0

115, 127, 83(108 ± 22.7)

11, 9, 13( 11 ± 2.0)

21, 23, 26( 23 ± 2.5)

21, 18, 22( 20 ± 2.1)

8, 7, 7( 7 ± 0.6)

15.6

97, 81, 93( 90 ± 8.3)

ND

ND

ND

ND

31.3

84, 83, 79( 82 ± 2.6)

17, 8, 21( 15 ± 6.7)

ND

20, 22, 28( 23 ± 4.2)

7, 6, 8( 7 ± 1.0)

62.5

82, 79, 80( 80 ± 1.5)

8, 7, 27( 14 ± 11.3)

ND

16, 28, 22( 22 ± 6.0)

2, 11, 7( 7 ± 4.5)

125

87, 83, 85( 85 ± 2.0)

11, 12, 15( 13 ± 2.1)

20, 12, 19( 17 ± 4.4)

13, 18, 19( 17 ± 3.2)

10, 6, 1( 6 ± 4.5)

250

58, 38, 55( 50 ± 10.8)

18, 8, 12( 13 ± 5.0)

22, 19, 26( 22 ± 3.5)

22, 23, 18( 21 ± 2.6)

8, 7, 7( 7 ± 0.6)

500

40*, 41*, 51*( 44 ± 6.1)

19, 3, 7( 10 ± 8.3)

14, 18, 24( 19 ± 5.0)

19, 18, 20( 19 ± 1.0)

5*, 8*, 3*( 5 ± 2.5)

1000

1*, 0*, 0*( 0 ± 0.5)

23, 21, 15( 20 ± 4.2)

14*, 11*, 17*( 14 ± 3.0)

1*, 1*, 7*( 3 ± 3.5)

2000

2, 4, 6( 4 ± 2.0)

4000

0*, 0*, 0*( 0 ± 0.0)

S9 mix(+)

0

123, 144, 155(141 ± 16.3)

7, 12, 10( 10 ± 2.5)

30, 18, 23( 24 ± 6.0)

37, 41, 32( 37 ± 4.5)

12, 14, 15( 14 ± 1.5)

15.6

103, 114, 121( 113 ± 9.1)

ND

ND

ND

ND

31.3

81, 117, 111( 103 ± 19.3)

ND

ND

ND

ND

62.5

93, 95, 105( 98 ± 6.4)

9, 14, 13( 12 ± 2.6)

ND

26, 35, 22( 28 ± 6.7)

7, 16, 12( 12 ± 4.5)

125

96, 98, 93( 96 ± 2.5)

17, 10, 8( 12 ± 4.7)

29, 21, 18( 23 ± 5.7)

26, 22, 25( 24 ± 2.1)

7, 6, 14( 9 ± 4.4)

250

56, 71, 83( 70 ± 13.5)

10, 10, 7( 9 ± 1.7)

32, 26, 22( 27 ± 5.0)

21, 27, 28( 25 ± 3.8)

9, 10, 15( 11 ± 3.2)

500

45*, 57*, 68*( 57 ± 11.5)

7, 4, 7( 6 ± 1.7)

26, 12, 19( 19 ± 7.0)

25, 18, 22( 22 ± 3.5)

10, 8, 4( 7 ± 3.1)

1000

4, 1, 3( 3 ± 1.5)

20, 23, 29( 24 ± 4.6)

20, 14, 23( 19 ± 4.5)

5, 8, 7( 7 ± 1.5)

2000

0*, 0*, 1*( 0 ± 0.6)

15, 12, 6( 11 ± 4.6)

0*, 0*, 0*( 0 ± 0.0)

0*, 0*, 0*( 0 ± 0.0)

4000

 

 

0*, 0*, 0*( 0 ± 0.0)

 

 

PositivecontrolS9 mix (-)

Chemical

AF2

SA

AF2

AF2

9AA

Dose(μg/plate)

0.01

0.5

0.01

0.1

80

Number ofcolonies/plate

591, 481, 463(512 ± 69.3)

263, 262, 225(250 ± 21.7)

108, 121, 101(110 ± 10.1)

785, 800, 745(777 ± 28.4)

1064,1104,971(1064±68.2)

PositivecontrolS9 mix (+)

Chemical

2AA

2AA

2AA

2AA

2AA

Dose(μg/plate)

1

2

10

0.5

2

Number ofcolonies/plate

2008,1637,1544(1730±245.5)

212, 229, 230( 224 ± 10.1)

1166,1335,1277(1259±85.9)

573, 498, 438(503 ± 67.6)

262, 217, 221( 233 ± 24.9)

AF2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, SA: Sodium azide, 9AA: 9-Aminoacridine, 2AA: 2-Aminoanthracene

*: Inhibition was observed against growth of the bacteria.

**: Purity was above 95% and impurity was unknown.

ND: Not done

Table 2 Mutagenicity of 2-hydroxybenzaldehyde** in reverse mutation test (II) on bacteria

With(+) orwithout(-)S9 mix

Test substancedose(μg/plate)

Number of revertants (Number of colonies/plate, Mean ±S.D.)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

S9 mix(-)

0

102, 98, 89( 96 ± 6.7)

16, 13, 12( 14 ± 2.1)

23, 22, 21( 22 ± 1.0)

27, 19, 27( 24 ± 4.6)

5, 11, 10( 9 ± 3.2)

15.6

82, 74, 82( 79 ± 4.6)

ND

ND

ND

ND

31.3

88, 92, 99( 93 ± 5.6)

10, 8, 10( 9 ± 1.2)

ND

21, 28, 28( 26 ± 4.0)

12, 7, 10( 10 ± 2.5)

62.5

75, 74, 82( 77 ± 4.4)

15, 10, 11( 12 ± 2.6)

ND

31, 30, 35( 32 ± 2.6)

4, 11, 8( 8 ± 3.5)

125

96, 64, 78( 79 ± 16.0)

11, 10, 13( 11 ± 1.5)

31, 31, 23( 28 ± 4.6)

27, 23, 31( 27 ± 4.0)

10, 4, 7( 7 ± 3.0)

250

66, 50, 59( 58 ± 8.0)

6, 6, 5( 6 ± 0.6)

31, 24, 38( 31 ± 7.0)

22, 23, 11( 19 ± 6.7)

7, 3, 2( 4 ± 2.6)

500

34*, 50*, 44*( 43 ± 8.1)

5, 7, 3( 5 ± 2.0)

20, 12, 28( 20 ± 8.0)

21, 23, 23( 22 ± 1.2)

4, 12, 8( 8 ± 4.0)

1000

3*, 0*, 2*( 2 ± 1.5)

23, 21, 25( 23 ± 2.0)

11*, 19*, 17*( 16 ± 4.2)

6*, 5*, 7*( 6 ± 1.0)

2000

8, 9, 8( 8 ± 0.6)

4000

0*, 0*, 0*( 0 ± 0.0)

S9 mix(+)

0

100, 112, 109(107 ± 6.2)

14, 10, 8( 11 ± 3.1)

30, 25, 29( 28 ± 2.6)

40, 31, 37( 36 ± 4.6)

5, 18, 12( 12 ± 6.5)

15.6

89, 120, 104( 104 ±15.5)

ND

ND

ND

ND

31.3

115, 116, 105( 112 ± 6.1)

ND

ND

ND

ND

62.5

103, 76, 96( 92 ± 14.0)

10, 10, 11( 10 ± 0.6)

ND

29, 40, 29( 33 ± 6.4)

9, 5, 11( 8 ± 3.1)

125

94, 98, 88( 93 ± 5.0)

7, 19, 13( 13 ± 6.0)

37, 29, 37( 34 ± 4.6)

28, 38, 24( 30 ± 7.2)

7, 13, 11( 10 ± 3.1)

250

63, 49, 72( 61 ± 11.6)

20, 7, 3( 10 ± 8.9)

47, 42, 34( 41 ± 6.6)

28, 23, 27( 26 ± 2.6)

11, 13, 8( 11 ± 2.5)

500

41*, 49*, 52*( 47 ± 5.7)

8, 5, 5( 6 ± 1.7)

33, 24, 31( 29 ± 4.7)

27, 29, 20( 25 ± 4.7)

7, 7, 3( 6 ± 2.3)

1000

1*, 0*, 1*( 1 ± 0.6)

27, 29, 21( 26 ± 4.2)

30, 19, 20( 23 ± 6.1)

8, 8, 4( 7 ± 2.3)

2000

0*, 0*, 0*( 0 ± 0.0)

17, 8, 14( 13 ± 4.6)

17*, 13*, 9*( 13 ± 4.0)

0*, 0*, 0*( 0 ± 0.0)

4000

 

 

0*, 0*, 0*( 0 ± 0.0)

 

 

PositivecontrolS9 mix (-)

Chemical

AF2

SA

AF2

AF2

9AA

Dose(μg/plate)

0.01

0.5

0.01

0.1

80

Number ofcolonies/plate

535, 591, 587(571 ± 31.2)

267, 284, 275(275 ± 8.5)

152, 146, 140(146 ± 6.0)

761, 734, 764(753 ± 16.5)

1132, 929, 1397(1153±234.7)

PositivecontrolS9 mix (+)

Chemical

2AA

2AA

2AA

2AA

2AA

Dose(μg/plate)

1

2

10

0.5

2

Number ofcolonies/plate

1464,1444,1498(1469±27.3)

291, 304, 309( 301 ± 9.3)

1422,1450,1427(1433±14.9)

342, 363, 371(359 ± 15.0)

263, 294, 277( 278 ± 15.5)

AF2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, SA: Sodium azide, 9AA: 9-Aminoacridine, 2AA: 2-Aminoanthracene

*: Inhibition was observed against growth of bacteria.

**: Purity was above 95% and impurity was unknown.

ND: Not done

Applicant's summary and conclusion

Conclusions:
2-Hydroxybenzaldehyde was not mutagenic to Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, either with or without an exogenous metabolic activation system.