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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation

Link to relevant study records
Reference
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:
10 Nov - 01 Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline-conform study under GLP without deviations
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
Identification:
Identification: Art. 818841 (1-Chlorpropane)
Batch: S4428641
Purity: 99.5% (GC, area%)
Appearance: Clear, colourless
Expiry Date: 31 August 2019
Storage Conditions: In the refrigerator (between 8 and 4°C)
Stability in Solvent: Not indicated by the Sponsor
Safety Precautions: Routine hygienic procedures will be sufficient to ensure personnel health and safety.


Target gene:
his and trp operon
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle:
Solvent used: DMSO
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and conditions:
METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 60 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
in strains TA1535 and TA100
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
the test item did not induce gene mutations
Additional information on results:
TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: not soluble
Precipitation: none
Other confounding effects:none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment I: TA 1535 with S9 mix: 5000 µg/plate
Experiment II: TA 100 with and without S9 mix: 5000 µg/plate

Summary of Experiment I

 

TestGroup

Dose Level

(per plate)

TA 1535

Revertant Colony Counts (Mean ±SD)

TA 1537

Revertant Colony Counts (Mean ±SD)

TA 98

Revertant Colony Counts (Mean ±SD)

TA 100

Revertant Colony Counts (Mean ±SD)

WP2 uvrA

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

Without

DMSO

 

19 ± 3

9 ± 2

21 ± 2

121 ± 3

30 ± 5

Activation

Untreated

 

20 ± 3

10 ± 1

23 ± 8

160 ± 23

34 ± 7

 

Art. 818841

3 µg

23 ± 4

8 ± 1

23 ± 3

150 ± 10

34 ± 3

 

(1-Chlorpropane)

10 µg

24 ± 1

11 ± 4

24 ± 1

140 ± 4

39 ± 7

 

 

33 µg

20 ± 4

12 ± 4

21 ± 6

142 ± 32

33 ± 6

 

 

100 µg

22 ± 4

7 ± 2

28 ± 7

136 ± 13

33 ± 6

 

 

333 µg

25 ± 6

13 ± 2

30 ± 4

133 ± 16

36 ± 5

 

 

1000 µg

18 ± 3

9 ± 3

20 ± 1

125 ± 17

33 ± 6

 

 

2500 µg

22 ± 8

12 ± 2

23 ± 7

118 ± 3

33 ± 4

 

 

5000 µg

24 ± 5

8 ± 2

27 ± 5

107 ± 3

39 ± 8

 

NaN3

10 µg

1409 ± 93

 

 

2002 ± 71

 

 

4-NOPD

10 µg

 

 

398 ± 34

 

 

 

4-NOPD

50 µg

 

86 ± 6

 

 

 

 

MMS

2.0 µL

 

 

 

 

921 ± 43

 

 

 

 

 

 

 

 

With

DMSO

 

15 ± 6

15 ± 2

36 ± 3

115 ± 3

46 ± 9

Activation

Untreated

 

11 ± 1

13 ± 3

42 ± 4

159 ± 11

51 ± 6

 

Art. 818841

3 µg

13 ± 3

17 ± 4

34 ± 11

121 ± 20

46 ± 7

 

(1-Chlorpropane)

10 µg

15 ± 0

15 ± 2

33 ± 8

129 ± 3

37 ± 4

 

 

33 µg

15 ± 4

12 ± 3

32 ± 10

121 ± 13

47 ± 7

 

 

100 µg

12 ± 2

13 ± 2

29 ± 7

136 ± 16

52 ± 10

 

 

333 µg

11 ± 1

13 ± 4

31 ± 8

117 ± 19

47 ± 10

 

 

1000 µg

11 ± 2

15 ± 1

32 ± 5

128 ± 15

53 ± 11

 

 

2500 µg

14 ± 2

12 ± 1

26 ± 1

106 ± 11

43 ± 7

 

 

5000 µg

7 ± 1

11 ± 1

24 ± 7

90 ± 17

51 ± 8

 

2-AA

2.5 µg

381 ± 23

139 ± 12

3212 ± 220

3893 ± 272

 

 

2-AA

10.0 µg

 

 

 

 

365 ± 15

Summary of Experiment II

Metabolic

Activation

 

 

TA 1535

Revertant Colony Counts (Mean ±SD)

TA 1537

Revertant Colony Counts (Mean ±SD)

TA 98

Revertant Colony Counts (Mean ±SD)

TA 100

Revertant Colony Counts (Mean ±SD)

WP2 uvrA

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

Without

DMSO

 

11 ± 5

8 ± 3

25 ± 3

112 ± 19

31 ± 6

Activation

Untreated

 

12 ± 3

10 ± 0

22 ± 2

172 ± 8

43 ± 9

 

Art. 818841

33 µg

10 ± 2

10 ± 4

19 ± 2

124 ± 19

38 ± 11

 

(1-Chlorpropane)

100 µg

10 ± 1

10 ± 1

21 ± 2

111 ± 2

39 ± 9

 

 

333 µg

10 ± 4

9 ± 1

24 ± 5

90 ± 9

41 ± 10

 

 

1000 µg

11 ± 1

10 ± 3

15 ± 2

67 ± 6

33 ± 1

 

 

2500 µg

12 ± 4

8 ± 5

14 ± 5

51 ± 3

30 ± 8

 

 

5000 µg

6 ± 1

10 ± 4

19 ± 3

49 ± 7

27 ± 8

 

NaN3

10 µg

1268 ± 104

 

 

1898 ± 68

 

 

4-NOPD

10 µg

 

 

479 ± 44

 

 

 

4-NOPD

50 µg

 

103 ± 9

 

 

 

 

MMS

2.0 µL

 

 

 

 

812 ± 40

 

 

 

 

 

 

 

 

With

DMSO

 

12 ± 4

10 ± 1

34 ± 2

101 ± 10

45 ± 2

Activation

Untreated

 

8 ± 1

14 ± 3

33 ± 9

154 ± 13

46 ± 3

 

Art. 818841

33 µg

12 ± 5

9 ± 2

38 ± 5

99 ± 3

45 ± 4

 

(1-Chlorpropane)

100 µg

9 ± 3

11 ± 5

33 ± 7

89 ± 5

38 ± 3

 

 

333 µg

13 ± 6

10 ± 4

28 ± 8

74 ± 7

40 ± 2

 

 

1000 µg

7 ± 2

7 ± 3

37 ± 9

67 ± 12

44 ± 7

 

 

2500 µg

8 ± 2

7 ± 3

28 ± 6

51 ± 13

33 ± 3

 

 

5000 µg

7 ± 1

10 ± 1

32 ± 2

43 ± 10

29 ± 4

 

2-AA

2.5 µg

340 ± 26

128 ± 9

2717 ± 489

2609 ± 443

 

 

2-AA

10.0 µg

 

 

 

 

317 ± 16

 

 

 

 

 

 

 

 

Key to Plate Postfix Codes:                     

P: Precipitate

M: Manuel Count

R:  Reduced Background growth

D:  Densely Colored Plate

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The test item Art. 818841 (1-Chlorpropane) was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:   3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                            33; 100; 333; 1000; 2500; and 5000 µg/plate

 

No precipitation of the test item occurred up to the highest investigated dose.

 

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

 

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain

Experiment I

 

Experiment II

 

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

/

5000

/

/

TA 1537

/

/

/

/

TA 98

/

/

/

/

TA 100

/

/

5000

5000

WP2 uvrA

/

/

/

/

/ = no toxic effect (induction factor >0.5)

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Art. 818841 (1-Chlorpropane) at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in bacteria

Mutagenicity of 1-chloropropane was tested in a GLP-compliant bacterial reverse mutation assay according to GLP and OECD TG 471 performed with a standard battery of Salmonella typhimurium tester strains including TA 1535, TA 1537, TA 98 and TA 100 and WP2uvrA bacterial cells (reference 7.6.1-1). Based on the results of a preliminary cytotoxicity test, the highest concentration of 5000 µg/plate was tested in all strains. 1-chloropropane did not exhibit mutagenic properties in the absence or presence of metabolic activation. Toxicity indicated by reduced number of revertants was observed at the highest concentration tested in the presence of metabolic activation for TA 1535 and in the absence and presence of metabolic activation for TA 100. Positive control substances induced a distinct increase in the number of revertants in all strains with and without metabolic activation thereby proving validity of the assay. Based on the results of the conducted study, 1-chloropropane is not considered to exhibit mutagenic properties in bacterial cells.

Justification for classification or non-classification

Based on the available data on genetic toxicity, there is no indication that 1-chloropropane induces genetic toxicity in bacteria. However, no final decision on classification for genetic toxicity according to Regulation (EC) 1272/2008 can be made, as no information on chromosomal aberration and mutagenicity in mammalian cells/in vivo is available.