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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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
Study period:
18 September 1996 to 25 November 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

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:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MOHW (Pharmaceutical Affairs Bureau Notification No. 24 September 11, 1989)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
liquid: viscous
Details on test material:
- Physical state: brown viscous liquid.
- Purity: 100% as manufactured; 45% w/w in highly refined base oil

Method

Target gene:
- S. typhimurium: Histidine synthesis.
- E. coli: Tryptophan synthesis.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: The broth used to grow overnight cultures of tester strains was Vogel-Bonner salt solution supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2. Bottom agar (25 mL per 15 x 100 mm petri dish) was added to the Vogel-Bonner minimal medium E and supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose. Top agar consisting of 0.7 % (w/v) agar and 0.5 % (w/v) sodium chloride and was supplemented with 10 mL of 0.5 mM histidine/ biotin solution per 100 mL agar. When S9 mix was required, 2.0 mL of the supplemented top agar was used in the overlay. However, when S9 was not required, water was added to the supplemented top agar (0.5 mL of water per 2 mL of supplemented top agar) and the resulting 2.5 mL of diluted supplemented top agar was used for the overlay.
- Properly maintained: yes.
- Periodically checked for genotype stability: Yes. The presence of the pKM101 plasmid was confirmed for tester strains TA98 and TA100 by demonstration of resistance to ampicillin (10 µg). The presence of the rfa wall mutation was confirmed by testing the sensitivity of each culture to crystal violet (10 µg). The presence of the uvrB excision repair deletion was confirmed by demonstration of the sensitivity of cultures to UV light.
- Periodically "cleansed" against high spontaneous background: Yes. The mean numbers of spontaneous revertants per plate in the vehicle controls that are characteristic of the respective strains were demonstrated by plating 100 µg aliquots of the culture along with the appropriate vehicle on selective media.
Additional strain / cell type characteristics:
other: rfa and uvrB mutations in all strains and pKM101 mutation in TA98 and TA100.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: The broth used to grow overnight cultures of tester strains was Vogel-Bonner salt solution supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2. Bottom agar (25 mL per 15 x 100 mm petri dish) was added to the Vogel-Bonner minimal medium E and supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose. Top agar consisting of 0.7 % (w/v) agar and 0.5 % (w/v) sodium chloride and was supplemented with 10 mL of 0.5mM tryptophan solution per 100 mL agar. When S9 mix was required, 2.0 mL of the supplemented top agar was used in the overlay. However, when S9 was not required, water was added to the supplemented top agar (0.5 mL of water per 2 mL of supplemented top agar) and the resulting 2.5 mL of diluted supplemented top agar was used for the overlay.
- Properly maintained: yes.
- Periodically checked for genotype stability: Yes. The presence of the uvrA excision repair deletion was confirmed by demonstration of the sensitivity of cultures to UV light.
- Periodically "cleansed" against high spontaneous background: Yes. The mean numbers of spontaneous revertants per plate in the vehicle controls that are characteristic of the respective strains were demonstrated by plating 100 µg aliquots of the culture along with the appropriate vehicle on selective media.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
100, 250, 500, 1000, 5000, 10000 µg/plate (all tester strains investigated both in the presence and absence of metabolic activation).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Pluronic F127 (25% w/w in ethanol).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Pluronic F127 (25% w/w in ethanol)
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene; ICR-191
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation).

DURATION
- Exposure duration: plates were inverted and incubated for 48 ± 8 hours at 37 ± 2 ºC.

NUMBER OF REPLICATIONS: test concentrations were performed in triplicate.

DETERMINATION OF CYTOTOXICITY
The condition of the bacterial background lawn was evaluated for evidence of cytotoxicity and test material precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate.

COLONY COUNTING
- The numbers of revertant colonies per plate for the vehicle and negative controls and all plates containing test material were counted manually. The numbers of revertant colonies per plate for positive controls were counted by automated colony counter.

STERILITY CONTROL
- Test solution sterility: The highest test material concentration was plated on agar without the addition of a culture, to assess the sterility of the test system.
- S9 mix sterility: The S9 mix was assessed for sterility by plating 0.5 mL on selective agar.

PRELIMINARY STUDY
- The cytotoxicity of the test material to the test system was determined in order to select an appropriate dosing regimen for the definitive study. The test was performed using strains TA100 and WP2uvrA both in the presence and absence of 10% S9 mix at ten concentrations between 10.0 and 10,000 µg/plate.
Evaluation criteria:
Test data from individual experiments was considered valid if:
a) all tester strain cultures exhibit sensitivity to UV light,
b) all Salmonella cultures exhibit sensitivity to crystal violet,
c) cultures of tester strains TA98 and TA100 exhibit resistance to ampicillin,
d) all tester strain cultures exhibit a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions,
e) positive controls must exhibit a 3-fold increase over the value of the vehicle control for that strain
f) a minimum of three non-toxic doses are required to evaluate assay data.

Once the criteria for a valid assay have been met, responses observed in the assay are evaluated as follows:

Tester strains TA98, TA100 and WPuvrA
For a test material to be considered positive, it must produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test material.

Tester strains TA1535 and TA1537
For a test material to be considered positive, it must produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test material.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Test material handling
The test material was not soluble and did not produce a workable suspension in ethanol, DMSO, or acetone. A workable suspension was achieved using Pluronic F127 25% w/w in ethanol and hence it was selected as the vehicle in the study. At 200 mg/mL, which was the most concentrated stock solution prepared, the test material formed an opaque, yellow suspension. The test material remained in suspension in all succeeding dilutions prepared for the mutagenicity assay.

- Dose Range Finding Study
Under the conditions of the study, no cytotoxicity was observed up to 10,000 µg/plate in either strain tested, either in the presence or absence of metabolic activation as evidenced by a normal background lawn and no reduction in the number of revertants per plate. Test material precipitate was observed on the plates at 667 µg/plate and above with tester strain TA100 in both the presence and absence of S9 mix and with tester strain WP2uvrA in the absence of S9 mix. With tester strain WP2uvrA in the presence of S9 mix test material precipitate was observed on the plates at 333 µg/plate and above.

- S9 Optimisation Study
Under the conditions of the study, no effect was observed due to varying the S9 homogenate concentration in this experiment. In the absence of an effect, a 10% S9 mix was used in the definitive mutagenicity assay.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenicity Assay

In the initial mutagenicity assay, and in the confirmatory mutagenicity assay, all data were acceptable and no positive increases in the number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. No cytotoxicity was observed up to 10,000 µg/plate with any of the tester strains in either the presence or absence of S9 mix. Test material precipitate was observed on plates dosed at 1,000 µg/plate and above. All validity criteria for the study were met.

Table 1: Mutagenicity Assay Results Summary (mean values)

With S9

Dose (µg)/plate

TA98 (SD)

TA100 (SD)

TA1535 (SD)

TA1537 (SD)

WP2uvrA- (SD)

Background lawn

Vehicle control

30 (7)

109 (15)

15 (6)

9 (2)

14 (6)

1

Test material

100

35 (4)

113 (11)

18(2)

7 (2)

13 (4)

1

250

24 (4)

101 (15)

15 (2)

8 (2)

10 (4)

1

500

39 (10)

110 (25)

18 (5)

8 (4)

14 (4)

1

1000

31 (7)

118 (9)

14 (4)

11 4()

18 (3)

1 sp

5000

19 (4)

130 (15)

14 (4)

4 (1)

8 (2)

1 hp*

10000

23 (6)

123 (30)

8 (4)

6 (4)

10 4()

1 hp*

Positive control

196 (36)

1025 (168)

144 (5)

94 (13)

327 (28)

1

Without S9

 

 

Vehicle control

18 (3)

94 (14)

12 (2)

7 (5)

12 (1)

1

Test material

100

20 (1)

77 (11)

13 (6)

7 (2)

15 (5)

1

250

17 (3)

93 (4)

13 (4)

6 (1)

12 (6)

1

500

27 (4)

93 (12)

12 (2)

6 (1)

8 (2)

1

1000

26 (3)

81 (5)

18 (2)

6 (2)

11 (4)

1 sp

5000

12 (6)

93 (17)

9 (5)

5 (1)

9 (2)

1 hp*

10000

11 (4)

96 (5)

7 (2)

5 (2)

12 (5)

1 hp*

Positive control

151 (28)

780 (31)

581 (22)

381 (41)

181 (55)

1

Table 2: Confirmatory Mutagenicity Assay Results Summary (mean values)

With S9

Dose (µg)/plate

TA98 (SD)

TA100

(SD)

TA1535 (SD)

TA1537 (SD)

WP2uvrA- (SD)

Background lawn

Vehicle control

32 (2)

118 (17)

14 (5)

10 (4)

11 (1)

1

Test material

100

28 (9)

135 (15)

21 (5)

8 (1)

19 (6)

1

250

36 (2)

136 (5)

10 (2)

7 (0)

13 (4)

1

500

25 (4)

131 (11)

15 (4)

9 (2)

13 (2)

1

1000

40 (5)

118 (20)

15 (3)

8 (2)

12 (5)

1 sp

5000

31 (7)

140 (8)

9 (1)

9 (5)

9 (4)

1 hp*

10000

26 (6)

152 (11)

10 (1)

5 (2)

10 (1)

1 hp*

Positive control

258 (21)

1452 (49)

179 (11)

185 (9)

259 (99)

1

Without S9

 

 

Vehicle control

21 (7)

98 (1)

13 (3)

6 (6)

14 (4)

1

Test material

100

22 (10)

106 (6)

12 (8)

7 (3)

13 (5)

1

250

23 (4)

106 (4)

14 (3)

4 (3)

12 (2)

1

500

19 (6)

114 (12)

10 (3)

3 (2)

14 (6)

1

1000

17 (3)

114 (11)

12 (2)

5 (3)

11 (4)

1 sp

5000

18 (8)

116 (13)

8 (3)

3 (2)

8 (2)

1 hp*

10000

11 (4)

115 (23)

8 (3)

4 (3)

8 (1)

1 hp*

Positive control

211 (40)

733 (50)

725 (11)

934 (42)

361 (22)

1

sp = slight precipitate, hp = heavy precipitate

* a dissecting microscope was used to aid in scoring revertants and reviewing plates due to the presence of large amounts of test material precipitate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the study, in both an initial and confirmatory assay, the test material did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver (S9). No cytotoxicity was observed up to 10,000 µg/plate in any of the strains tested either in the presence or absence of S9 mix. Test material precipitate was observed on the plates at concentrations of 1000 µg/plate and above. The study is considered to be reliable, relevant and adequate for risk assessment and for classification and labelling purposes.
Executive summary:

The potential of the test material to cause gene mutation in bacterial strains was determined according to the following guidelines, OECD 471, EPA OPPTS 870.5100 and EU Method B.13/14. During the study, four strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and one strain of Escherichia coli (WP2uvrA) were treated in the presence and absence of a rat liver derived metabolic activation system (S9 mix). In two separate assays, the test material did not induce any significant increases in the number of revertant colonies, in any of the tester strains, either in the presence or absence of S9 mix. Furthermore, no cytotoxicity was observed up to 10,000 µg per plate in any of the strains tested either in the presence or absence of S9 mix. Test material precipitate was observed on the plates at concentrations of 1000 µg per plate and above.