Registration Dossier

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:
22 Feb 2016 to 11 Apr 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted 21 Jul 1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(Official Journal of the European Union No. L142, 31 May 2008)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): MIPA-Cocoyl-Sarkosinat
- Substance type: organic (UVCB)
- Physical state: clear yellow-brown viscous liquid
- Analytical purity: 100%, according to definition of UVCB
- Impurities (identity and concentrations): no impurities, UVCB
- Lot/batch No.: 070715
- Expiration date of the lot/batch: 31 Dec 2016
- Stability under test conditions: stable in vehicle water
- Storage condition of test material: at room temperature
- Other: pH 5.7 - 6.0 (1% in water)

Method

Target gene:
His-operon, Trp-operon
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:
other: rfa, gal, chl, bio, uvrB
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9-mix (5 or 10%, depending on strain and experiment)
Test concentrations with justification for top dose:
Exp 1: 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate ± S9 (TA100, WP2uvrA); 5.4, 17, 52, 164, 512, 1600 µg/plate - S9, 17, 52, 164, 512, 1600 µg/plate + S9 (TA1535, TA1537, TA98)
Exp 2: 154, 275, 492, 878, 1568, 2800 µg/plate ± S9 (TA1535, TA1537, TA98, TA100); 492, 878, 1568, 2800, 5000 µg/plate ± S9 (WP2uvrA)
Exp 3: 48, 87, 154, 275, 492 µg/plate - S9 (TA1535, TA1537, TA98, TA100); 48, 87, 154, 275, 492 µg/plate + S9 (TA1537)
Exp 4: 48, 87, 154, 275, 492, 878 µg/plate - S9 (TA98)
Exp 5: 154, 275, 492, 878, 1568, 2800, 5000 µg/plate - S9 (TA98)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
other: - S9: sodium azide (TA1535, 5 µg/plate); ICR-191 (TA1537, 2.5 µg/plate); 2-nitrofluorene (TA98, 10 µg/plate); methylmethanesulfonate (TA100, 650 µg/plate); 4-nitroquinoline N-oxide (WP2uvrA, 10 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with S9-mix
Positive control substance:
other: + S9: 2-aminoanthracene (TA1535, 2.5 µg/plate; TA1537 [5% S9], 2.5 µg/plate; TA1537 [10% S9], 5 µg/plate; TA98, 1 µg/plate; TA100 [5% S9], 1 µg/plate; TA 100 [10% S9], 2 µg/plate; WP2uvrA, 15 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 h

SELECTION AGENT (mutation assays): culture plates lacking histidine (Salmonella) or tryptophan (E. coli)

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: reduction of revertant numbers, reduction of background lawn, size increase of microcolonies

OTHER:
In the combined range finder/experiment 1 a concentration of 5% S9-mix was used for metabolical activation. In the repeat experiments an S9-mix concentration of 10% was chosen, which is considered to be an appropriate variation of experimental parameters for repeat experiments, according to the experience of the testing laboratory.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
Acceptability criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected concentration range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

DATA EVALUATION
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strains TA100 or WP2uvrA is not greater than two (2) times the revertant number in the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the revertant number in the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strains TA100 or WP2uvrA is greater than two (2) times the revertant number in the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the revertant number in the concurrent vehicle control.
b) In case a follow-up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow-up experiment.
Statistics:
None

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
Range finder was performed in TA100 and WP2uvrA, which was reported within the scope of Experiment 1.

COMPARISON WITH HISTORICAL CONTROL DATA:
The observed results were in accordance with the laboratory historical control data ranges, indicating that the test conditions were adequate and and that the metabolic activation system functioned properly. The only deviation from the historical control data ranges was observed for the response of WP2uvrA (+ S9) in the second experiment, where the observed numbers of revertants exceeded the historical control data ranges. The positive control serves as a reference for the test system where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values and even exceeded the historical control range, this deviation in the mean plate count of the positive control did not affect the integrity of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Exp 1 (µg/plate): - S9: TA100 ≥512, WP2uvrA =5000, TA1535 ≥1600, TA1537 ≥512, TA98 ≥1600; + S9: TA100 ≥1600, TA1535 ≥512, TA1537 ≥1600, TA98 ≥ 1600
Exp 2: ± S9: No cytotoxicity
Exp 3 (µg/plate): - S9: TA1535≥492, TA1537≥492, TA100 ≥492; +S9: TA1537 ≥275
Exp 4 + 5 (µg/plate): - S9: TA98 ≥878
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment 1 (incl. results of dose range finding test with TA100 and WP2uvrA)

Dose (µg/plate)

Mean number of revertants ± SD

TA100

WP2uvrA

TA1535

TA1537

TA98

Without S9-mix

Pos. control

1094 ± 67

1614 ± 69

916 ± 16

678 ± 31

1621 ± 55

Solv. control

136 ± 6

16 ± 4

8 ± 6

5 ± 1

16 ± 2

1.7

134 ± 6

17 ± 2

---

---

---

5.4

120 ± 9

20 ± 7

9 ± 4

6 ± 5

13 ± 6

17

118 ± 8

16 ± 6

7 ± 4

4 ± 2

17 ± 5

52

103 ± 20

27 ± 6

4 ± 1

3 ± 1

17 ± 4

164

115 ± 9 n

20 ± 4

6 ± 3

4 ± 2 n

11 ± 2

512

39 ± 7 m

20 ± 2

2 ± 1

1 ± 1 s

11 ± 1

1600

e MC

15 ± 3

NP e MC

NP e MC

6 ± 2NP n

5000

0 ± 0a NP

10 ± 2n NP

---

---

---

With S9-mix (5%)

Pos. control

1404 ± 107

578 ± 49

325 ± 34

451 ± 85

1320 ± 28

Solv. control

124 ± 6

21 ± 9

6 ± 2

5 ± 2

23 ± 2

1.7

139 ± 2

29 ± 7

---

---

---

5.4

130 ± 19

34 ± 7

---

---

---

17

142 ± 12

19 ± 3

7 ± 3

3 ± 2

19 ± 1

52

128 ± 12

27 ± 7

9 ± 2

3 ± 3

23 ± 4

164

138 ± 14

34 ± 11

2 ± 1n

2 ± 2

14 ± 3

512

104 ± 14n

35 ± 11

1 ± 1s

1 ± 1n

14 ± 3

1600

9 ± 4m

21 ± 3

3 ± 2NP m

NP e MC

8 ± 2NP n

5000

0 ± 0a NP

14 ± 5n NP

---

---

---

MC: Microcolonies
NP: No precipitate
a: Bacterial background lawn absent
e: Bacterial background lawn extremely reduced
m: Bacterial background lawn moderately reduced
n: Normal bacterial background lawn
s: Bacterial background lawn slightly reduced
---: not tested

 

Table 2: Experiment 2

Dose (µg/plate)

Mean number of revertants ± SD

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Pos. control

814 ± 31

1005 ± 133

1862 ± 314

746 ± 44

538 ± 10

Solv. control

9 ± 3

4 ± 2

14 ± 4

94 ± 13

30 ± 4

154

4 ± 3

2 ± 1

10 ± 5

105 ± 6

---

275

3 ± 2n

1 ± 1n

9 ± 2n

56 ± 5n

---

492

0 ± 1m

e MC

5 ± 1m

e MC

24 ± 1

878

e NP MC

e MC

e MC

e MC

19 ± 3

1568

e NP MC

e MC

e MC

e MC

28 ± 6

2800

e NP MC

e NP MC

e NP MC

e NP MC

34 ± 11

5000

---

---

---

---

32 ± 4n NP

With S9-mix (10%)

Pos. control

173±16

411±52

657±75

1287±119

1500±104

Solv. control

5±2

4±1

13±1

88±19

15±5

154

4±1

4±3

20±3

94±1

---

275

3±1

1±1n

13±3

99±7n

---

492

2±1n

1±0s

15±3

86±22s

16±1

878

1±1s

e MC

11±2

e MC

18±2

1568

0±1m

e MC

8±2n

e MC

12±5

2800

e NP MC

e NP MC

1± 2s NP

e NP MC

13±8

5000

---

---

---

---

12±8n NP

MC: Microcolonies
NP: No precipitate
e: Bacterial background lawn extremely reduced
m: Bacterial background lawn moderately reduced
n: Normal bacterial background lawn
s: Bacterial background lawn slightly reduced
---: not tested

 

Table 3: Experiment 3

Dose (µg/plate)

Mean number of revertants ± SD

TA1535

TA1537

TA98

TA100

Without S9-mix

Pos. control

1060 ± 79

880 ± 265

1827 ± 118

1005 ± 33

Solv. control

6 ± 3

6 ± 2

18 ± 4

96 ± 16

48

11 ± 2

8 ± 4

20 ± 13

111 ± 9

87

6 ± 3

6 ± 2

21 ± 10

89 ± 5

154

5 ± 2

8 ± 3

19 ± 3

83 ± 22

275

6 ± 4n

5 ± 2n

59 ± 9

68 ± 15

492

1 ± 2s NP

2 ± 1s NP

42 ± 23n NP

11 ± 8n NP

With S9-mix (10%)

Pos. control

---

136 ± 12

---

---

Solv. control

---

12 ± 3

---

---

48

---

11 ± 6

---

---

87

---

9 ± 2

---

---

154

---

10 ± 3

---

---

275

---

5 ± 4

---

---

492

---

3 ± 2n NP

---

---

NP: No precipitate
n: Normal bacterial background lawn
s: Bacterial background lawn slightly reduced
---: not tested

Table 4: Experiment 4 and 5

Dose (µg/plate)

Mean number of revertants ± SD

TA98 Exp. 4

TA98 Exp. 5

Without S9-mix

Pos. control

1830 ± 182

1684 ± 46

Solv. control

13 ± 4

16 ± 6

48

11 ± 3

---

87

13 ± 3

---

154

10 ± 5

12 ± 1

275

9 ± 3

14 ± 1

492

9 ± 3

13 ± 2

878

10 ± 4

8 ± 4n

1568

---

6 ± 1s

2800

---

4 ± 3m

5000

---

e NP MC

MC: Microcolonies
NP: No precipitate
e: Bacterial background lawn extremely reduced
m: Bacterial background lawn moderately reduced
n: Normal bacterial background lawn
s: Bacterial background lawn slightly reduced
---: not tested

 

CONCLUSION:

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative