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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. QA statement)
Remarks:
Envigo, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD UK
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
solid
Details on test material:
Physical state/Appearance: Off-white solid
Batch: 0000149116
Purity: >80%
Expiry Date: 28 October 2017
Storage Conditions: Room temperature in the dark

Method

Target gene:
his
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 metabolic fraction, 10%
Test concentrations with justification for top dose:
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 microgram/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
Tetrahydrofuran
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other:
Details on test system and experimental conditions:
Top agar was prepared using 0.6% Bacto agar (lot number 6147883 03/21) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 hours.

Both plate incorporation method and pre-incubation methods were used in this study.

All of the plates were incubated at 37 ± 3 degrees C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).




Evaluation criteria:
The criteria for determining a positive result include any, one, or all of the following:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
E. coli WP2 uvr A
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
Additional information on results:
A test item precipitate (white and particulate in appearance) was noted at and above 500 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) or in Experiment 2 (preincubation method).

A small, statistically significant increase in TA98 revertant colony frequency was observed in the absence of S9-mix at 500 µg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 500 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.4 times the concurrent vehicle control.

Any other information on results incl. tables

Experiment 1- Without metabolic activation (Plate Incorporation)

 

Test Period

From: 10 March 2017- 16 March 2017

To: 13 March 2017- 19 March 2017

 

 

 

 

 

 

 

 

 

 

 

 

S9-Mix (-)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-Pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (THF)

120

101 (113)

118 10.4#

14

15 (13)

10 2.6

38

41 (38)

34 3.5

24

23 (27)

35 6.7

21

12 (15)

12 5.2

1.5μg

127

117 (123)

125 5.3

23

14 (17)

13 5.5

37

48 (44)

48 6.4

23

31 (31)

40 8.5

15

9 (14)

18 4.6

5μg

125

118 (117)

108 8.5

12

18 (15)

15 3.0

43

45 (42)

38 3.6

35

33 (34)

35 3.5

11

17 (14)

15 3.1

15μg

111

110 (115)

123 7.2

23

17 (19)

16 3.8

40

37 (35)

29 5.7

34

34 (36)

40 3.5

22

15 (17)

14 4.4

50μg

110

125 (124)

137 13.7

8

9 (9)

10 1.0

46

31 (38)

37 7.5

35

32 (33)

33 1.5

12

12 (14)

19 4.0

150μg

116

114 (120)

130 8.7

17

14 (14)

12 2.5

33

39 (36)

37 3.1

30

40 (34)

33 5.1

21

13 (17)

17 4.0

500μg

137P

122P (124)

112P 13.5

13P

14P (14)

16P 1.5

46 P

34 P (36)

28 P 3.1

39P *

39P (38)

36P 4.2

20P

15P (17)

17P 2.5

1500μg

115P

129P (123)

126P 7.4

10P

10P (10)

10P 0.0

32 P

24 P (36)

31 P 9.2

37P

31P (36)

39P 1.7

15P

12P (14)

14P 1.5

5000μg

116P

113P (110)

101P 7.9

13P

11P (12)

11p 1.2

30 P

14 P (26)

33 P 10.2

37P

31P (34)

31P 4.6

12P

15P (11)

7P 4.0

Positive controls S9- Mix (-)

Name Dose Level No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3μg

5μg

2μg

0.2μg

80μg

562

580 (594)

640 40.8

361

430 (396)

397 34.5

972

1028 (997)

992 28.4

108

110 (114)

124 8.7

211

295 (274)

315 55.2

( ) concurrent negative controls

# standard deviation

* P </= 0.05

Experiment 1- With Metabolic Activation (Plate Incorporation)

Test Period

From: 10 March 2017- 16 March 2017

To: 13 March 2017- 19 March 2017

 

 

 

 

 

 

 

 

 

 

 

 

S9-Mix (-)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-Pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (THF)

114

116 (113)

109 3.6#

12

10 (12)

15 2.5

33

32 (34)

37 2.6

23

23 (25)

28 2.9

9

18 (14)

14 4.5

1.5μg

99

99 (104)

113 8.1

7

13 (10)

11 3.1

35

42 (35)

28 7.0

39

29 (32)

29 5.8

14

10 (13)

14 4.5

5μg

103

106 (106)

110 3.5

11

11 (11)

12 0.6

28

27 (31)

37 5.5

28

23 (25)

25 2.5

14

8 (11)

12 3.1

15μg

107

124 (124)

142 17.5

11

12 (10)

8 2.1

24

41 (32)

31 8.5

33

36 (32)

28 4.0

18

15 (15)

11 3.5

50μg

121

135 (127)

126 7.1

11

12 (12)

13 1.2

27

29 (29)

31 2.0

21

27 (29)

38 8.6

9

18 (14)

16 4.7

150μg

132

129 (131)

131 1.5

11

19 (14)

12 4.4

29

31 (31)

33 2.0

21

28 (24)

22 3.8

15

14 (14)

13 1.0

500μg

115P

118P (119)

125P 5.1

19P

12P (11)

10P 1.2

37P

31P (33)

32P 3.2

18P

32P (30)

39P 10.7

15P

11P (13)

12P 2.1

1500μg

101P

134P (112)

101P 19.1

9P

14P (11)

10P 2.6

33P

19P (30)

38P 9.8

20P

27P (26)

32P 6.0

11P

9P (9)

7P 2.0

5000μg

117P

104P (109)

106P 7.0

12P

10P (12)

14P 2.0

33P

33P (30)

25P 4.6

26P

26P (24)

21P 2.9

10P

11P (10)

9P 1.0

Positive controls S9- Mix (-)

Name Dose Level No. of Revertants

2AA

2AA

2AA

BP

2AA

1μg

2μg

10μg

5μg

2μg

1940

1883 (1850)

1727 110.3

289

264 (264)

240 24.5

271

374 (347)

395 66.4

225

199 (217)

228 15.9

402

396 (416)

451 30.2

( ) Concurrent negative controls

# Standard deviation

P Precipitate

Experiment 2- Without metabolic activation (Pre-Incubation)

 

Test Period

From: 24 March

To: 27 March

 

 

 

 

 

 

 

 

 

 

 

 

S9-Mix (-)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-Pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (THF)

91

92 (89)

85 3.8#

14

9 (11)

11 2.5

35

37 (38)

43 4.2

13

21 (18)

20 4.4

8

13 (10)

8 2.9

15μg

89

86 (92)

102 8.5

8

8 (8)

9 0.6

32

28 (32)

36 4.0

15

21 (16)

12 4.6

4

8 (8)

11 3.5

50μg

83

78 (79)

73 4.0

8

10 (9)

9 1.0

33

42 (40)

44 5.9

18

14 (17)

19 2.6

11

5 (8)

8 3.0

150μg

83

88 (85)

85 2.5

9

9 (9)

8 0.6

40

36 (39)

41 2.6

21

22 (21)

19 15

8

10 (8)

7 1.5

500μg

101P

86P (93)

92P 7.5

14P

10P (12)

11P 2.1

40P

33P (37)

37P 3.5

16P

19P (20)

25P 4.6

12P

7P (9)

9P 2.5

1500μg

88P

96P (90)

86P 5.3

10P

12P (11)

11P 1.0

40P

35P (39)

42P 3.6

21P

19P (19)

17P 2.0

13P

11P (11)

8P 2.5

5000μg

94P

79P (88)

90P 7.8

9P

12P (10)

9P 1.7

34P

37P (34)

30P 3.5

21P

19P (21)

22P 1.5

10P

7P (10)

12P 2.5

Positive controls S9- Mix (-)

Name Dose Level No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3μg

5μg

2μg

0.2μg

80μg

511

542 (547)

588 38.7

193

190 (192)

193 1.7

283

248 (269)

276 18.5

190

191 (191)

192 1.0

387

213 (274)

223 97.7

( ) concurrent negative controls

# Standard deviation

P Precipitate

Experiment 2- With Metabolic Activation (Pre-Incubation)

Test Period

From: 24 March

To: 27 March

 

 

 

 

 

 

 

 

 

 

 

 

S9-Mix (-)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-Pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (THF)

96

95 (96)

98 1.5#

7

12 (12)

17 5.0

47

37 (37)

27 10.0

40

27 (28)

17 11.5

9

9 (8)

7 1.2

15μg

103

104 (103)

103 0.6

16

9 (13)

14 3.6

38

38 (38)

38 0.0

26

18 (28)

40 11.1

9

5 (9)

13 4.0

50μg

84

92 (89)

92 4.6

9

10 (11)

14 2.6

39

31 (36)

37 4.2

28

17 (24)

28 6.4

3

9 (7)

8 3.2

150μg

91

97 (94)

93 3.1

15

7 (12)

14 4.4

47

34 (42)

44 6.8

20

22 (24)

28 6.4

15

9 (10)

7 4.2

500μg

97P

91P (92)

88P 4.6

10P

12P (12)

14P 2.0

34P

45P (39)

39P 5.5

27P

30P (29)

30P 1.7

12P

8P (9)

7P 2.6

1500μg

101P

95P (96)

91P 5.0

11P

10P (11)

13P 1.5

36P

44P (40)

41P 4.0

29P

26P (26)

24P 2.5

10P

6P (9)

11P 2.6

5000μg

90P

97P (93)

93P 3.5

14P

12P (12)

11P 1.5

32P

39P (37)

41P 4.7

22P

23P (25)

29P 3.8

10P

9P (9)

7P 1.5

Positive controls S9- Mix (-)

Name Dose Level No. of Revertants

2AA

2AA

2AA

BP

2AA

1μg

2μg

10μg

5μg

2μg

1785

1819 (1725)

1570 135.0

270

276 (272)

270 3.5

257

209 (238)

248 25.5

120

108 (116)

120 6.9

248

271 (264)

272 13.6

( ) Concurrent negative controls

# Standard deviation

P Precipitate

Applicant's summary and conclusion

Conclusions:
Naturechem® GMHS (Glyceryl Monohydroxystearate) was non-mutagenic in the Ames Assay under the conditions of this test.