Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on results of the read across study, the test substance, 'Cocodimonium hydroxypropyl hydrolysed wool', is considered to be non-mutagenic with and without metabolic activation.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From September 20, 2017 to October 27, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (Presence and absence of S9)
Experiment 2: 0.15, 0.5, 1.5, 5, 15, 50, 150 500 μg/plate (Presence and absence of S9)
The maximum concentration was 5000 µg/plate (the maximum recommended dose level).
Vehicle / solvent:
In solubility checks performed in–house, the test substance was noted as fully soluble in sterile distilled water at 50 mg/mL. Sterile distilled water was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Identity: Sterile distilled water, Supplier: Aguettant, Batch number (purity): 3012486 (N/A), Expiry: 11/2018
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: Identity: 2-Aminoanthracene (2AA), CAS No.: 613-13-8, Batch number: STBB1901M9, Purity: 97.5%, Expiry date: 08 October 2019, Solvent: DMSO, Concentration: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 , 10 µg/plate for WP2uvrA
Details on test system and experimental conditions:
Bacteria
The five strains of bacteria used, and their mutations, are as follows:
1) Salmonella typhimurium
Strains - Genotype - Type of mutations indicated
TA1537 - his C 3076; rfa-; uvrB-: - frame shift
TA98 - his D 3052; rfa-; uvrB-; - R-factor
TA1535 - his G 46; rfa-; uvrB-: - base-pair substitution
TA100 - his G 46; rfa-; uvrB-;R-factor

2) Escherichia coli
Strain - Genotype - Type of mutations indicated
WP2uvrA - trp-; uvrA-: - base-pair substitution

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).

The bacteria used in the test were obtained from:
1) University of California, Berkeley, on culture discs, on 04 August 1995.
2) British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. 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 h. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
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 substance will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test substance activity. Results of this type will be reported as equivocal.
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. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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:
valid
Positive controls validity:
valid

Results

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test substance formulation was also shown to be sterile. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test substance, positive and vehicle controls, both with and without metabolic activation, are presented in Table 1 and Table 2 for Experiment 1 (plate incorporation) and Table 3 and Table 4 for Experiment 2 (pre-incubation).

Table 1: Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period

From: 09 October 2017
16 October 2017†

To: 12 October 2017
19 October 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

(Water)

103

90

106

(100)

8.5#

29

32

34

(32)

2.5

25

28

18

(24)

5.1

36

13

23

(24)

11.5

13

9

18

(13)

4.5

1.5 µg

103

92

109

(101)

8.6

27

28

28

(28)

0.6

27

26

18

(24)

4.9

25

36

24

(28)

6.7

21

10

15

(15)

5.5

5 µg

101

87

99

(96)

7.6

33

31

25

(30)

4.2

29

24

30

(28)

3.2

19

19

29

(22)

5.8

12

14

18

(15)

3.1

15 µg

96

98

92

(95)

3.1

29

29

26

(28)

1.7

29

21

34

(28)

6.6

29

19

16

(21)

6.8

12

20

16

(16)

4.0

50 µg

75

94

83

(84)

9.5

32

29

30

(30)

1.5

26

24

33

(28)

4.7

15

22

16

(18)

3.8

15

16

11

(14)

2.6

150 µg

8 S

10 S

18 S

(12)

5.3

15 S

12 S

17 S

(15)

2.5

24

17

19

(20)

3.6

11 S

5 S

5 S

(7)

3.5

4 S

4 S

9 S

(6)

2.9

500 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

8 S

20 S

20 S

(16)

6.9

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

1500 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

5000 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

466

611

578

(552)

76.0

1141

1211

1265

(1206)

62.2

757

858

930

(848)

86.9

206

193

227

(209)

17.2

435

497

498

(477)

36.1

Table 2: Test Results: Experiment 1 – With Metabolic Activation(Plate Incorporation)

Test Period

From: 09 October 2017
16 October 2017†

To: 12 October 2017
19 October 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

(Water)

106

87

89

(94)

10.4#

36

39

39

(38)

1.7

34

35

35

(35)

0.6

32

31

33

(32)

1.0

15

10

14

(13)

2.6

1.5 µg

69

81

76

(75)

6.0

42

45

37

(41)

4.0

38

36

41

(38)

2.5

33

23

28

(28)

5.0

9

19

15

(14)

5.0

5 µg

72

107

95

(91)

17.8

38

39

38

(38)

0.6

22

30

42

(31)

10.1

24

29

25

(26)

2.6

12

23

15

(17)

5.7

15 µg

104

68

120

(97)

26.6

43

39

41

(41)

2.0

23

37

39

(33)

8.7

35

38

25

(33)

6.8

18

14

14

(15)

2.3

50 µg

104

95

87

(95)

8.5

40

41

41

(41)

0.6

32

35

37

(35)

2.5

35

18

31

(28)

8.9

6

12

18

(12)

6.0

150 µg

73 S

52 S

63 S

(63)

10.5

38

44

43

(42)

3.2

34

28

24

(29)

5.0

23

23

23

(23)

0.0

9

12

16

(12)

3.5

500 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

27 S

20 S

28 S

(25)

4.4

0 V

0 V

0 V

(0)

0.0

3 S

3 S

9 S

(5)

3.5

1500 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

5000 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

2047

2156

2207

(2137)

81.7

385

377

326

(363)

32.0

601

512

560

(558)

44.5

248

285

260

(264)

18.9

465

475

437

(459)

19.7

Table 3: Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period

From: 24 October 2017

To: 27 October 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

(Water)

83

99

83

(88)

9.2#

14

8

11

(11)

3.0

21

22

37

(27)

9.0

22

17

18

(19)

2.6

6

12

12

(10)

3.5

0.15 µg

96

89

106

(97)

8.5

12

18

11

(14)

3.8

17

20

36

(24)

10.2

15

22

18

(18)

3.5

9

9

20

(13)

6.4

0.5 µg

106

103

105

(105)

1.5

20

10

11

(14)

5.5

19

25

17

(20)

4.2

18

25

14

(19)

5.6

13

17

8

(13)

4.5

1.5 µg

106

98

91

(98)

7.5

26

19

13

(19)

6.5

25

20

22

(22)

2.5

29

19

21

(23)

5.3

16

17

12

(15)

2.6

5 µg

94

97

104

(98)

5.1

11

12

11

(11)

0.6

23

23

24

(23)

0.6

15

13

13

(14)

1.2

18

14

6

(13)

6.1

15 µg

114

90

110

(105)

12.9

19

9

21

(16)

6.4

18

31

22

(24)

6.7

17

24

26

(22)

4.7

11

20

14

(15)

4.6

50 µg

76 S

89 S

91 S

(85)

8.1

6 S

18 S

13 S

(12)

6.0

31

16

18

(22)

8.1

15 S

20 S

10 S

(15)

5.0

12 S

4 S

9 S

(8)

4.0

150 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

21 S

12 S

21 S

(18)

5.2

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

500 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

786

849

829

(821)

32.2

1071

1118

1220

(1136)

76.2

833

777

715

(775)

59.0

208

233

200

(214)

17.2

171

209

185

(188)

19.2

Table 4: Test Results: Experiment 2 – With Metabolic Activation(Pre-Incubation)

Test Period

From: 24 October 2017

To: 27 October 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

(Water)

83

94

96

(91)

7.0#

13

18

8

(13)

5.0

33

33

39

(35)

3.5

21

17

26

(21)

4.5

13

14

15

(14)

1.0

0.15 µg

93

97

101

(97)

4.0

10

8

13

(10)

2.5

38

33

34

(35)

2.6

27

19

26

(24)

4.4

18

15

19

(17)

2.1

0.5 µg

98

93

99

(97)

3.2

13

11

12

(12)

1.0

42

36

38

(39)

3.1

37

12

20

(23)

12.8

25

14

8

(16)

8.6

1.5 µg

114

103

101

(106)

7.0

15

12

13

(13)

1.5

32

39

28

(33)

5.6

23

14

27

(21)

6.7

19

15

16

(17)

2.1

5 µg

88

73

103

(88)

15.0

13

8

9

(10)

2.6

31

22

37

(30)

7.5

36

22

25

(28)

7.4

20

6

17

(14)

7.4

15 µg

99

108

96

(101)

6.2

8

10

7

(8)

1.5

26

21

31

(26)

5.0

19

34

25

(26)

7.5

21

16

14

(17)

3.6

50 µg

116

93

82

(97)

17.3

15

9

9

(11)

3.5

31

25

22

(26)

4.6

32

25

31

(29)

3.8

17

19

16

(17)

1.5

150 µg

106 S

104 S

111 S

(107)

3.6

7 S

18 S

10 S

(12)

5.7

40

32

19

(30)

10.6

22

32

19

(24)

6.8

15

20

15

(17)

2.9

500 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

60 S

34 S

41 S

(45)

13.5

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1234

1172

1126

(1177)

54.2

222

237

255

(238)

16.5

259

202

186

(216)

38.4

117

114

149

(127)

19.4

329

288

299

(305)

21.2

†: Experimental procedure repeated at a later date due to high colony counts in the original test

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

S: Sparse bacterial background lawn

T: Toxic, no bacterial background lawn

V: Very weak bacterial background lawn

#: Standard deviation

The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) the test substance induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of metabolic activation (S9‑mix) initially from 150 µg/plate. Consequently, the toxic limit of the test substance was selected as the maximum dose level in the second mutation test. Results from the second mutation test showed that the test substance induced a stronger toxic response employing the pre‑incubation modification with weakened bacterial background lawns initially noted in the absence of S9‑mix from 50 µg/plate (all Salmonella strains) and 150 µg/plate (WP2uvrA). In the presence S9 -mix, weakened bacterial background lawns were initially noted from 150 µg/plate (TA100 and TA1535) and 500 µg/plate (TA98, TA1537 and WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9 -mix and experimental methodology. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9 -mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9 -mix) in Experiment 2 (pre‑incubation method). Minor statistical values were noted in Experiment 2 (TA100 at 0.5 and 15 µg/plate in the absence of S9 -mix), however these responses were within the in-house historical vehicle/untreated control values and were, therefore considered of no biological relevance. Based on the study results, the test substance was considered to be non-mutagenic with and without metabolic activation.

Conclusions:
Based on results of the read across study, the test substance is considered to be non-mutagenic with and without metabolic activation.
Executive summary:

A study was conducted to determine the genotoxic potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed silk' (active: 51%), using Ames test method,according to OECD Guideline 471, EU Method B13/14 and the USA, EPA OCSPP harmonized guideline, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the read across substance using both the Ames plate incorporation (Experiment 1) and pre-incubation (Experiment 2) methods at eight dose levels (i.e., 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation method) was predetermined and ranged from 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh read across substance formulations. The dose range was amended for Experiment 2, following the results of Experiment 1 and ranged from 0.15 to 500 µg/plate . There were eight read across substance concentrations(i.e., 0.15, 0.5, 1.5, 5, 15, 50, 150 500 μg/plate),which were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the toxic limit of the read across substance following the change in test methodology. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated. In the first mutation test (Experiment 1), the read across substance induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of metabolic activation (S9‑mix) initially from 150 µg/plate. Consequently, the toxic limit of the test substance was selected as the maximum dose level in the second mutation test (Experiment 2). Results from the second mutation test showed that the read across substance induced a stronger toxic response employing the pre‑incubation modification with weakened bacterial background lawns initially noted in the absence of S9‑mix from 50 µg/plate (all Salmonella strains) and 150 µg/plate (WP2uvrA). In the presence S9 -mix, weakened bacterial background lawns were initially noted from 150 µg/plate (TA100 and TA1535) and 500 µg/plate (TA98, TA1537 and WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the read across substance varied slightly between strain type, exposures with or without S9 -mix and experimental methodology. No read across substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the read across substance, either with or without metabolic activation (S9 -mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the read across substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). Minor statistical values were noted in Experiment 2 (TA100 at 0.5 and 15 µg/plate in the absence of S9 -mix), however these responses were within the in-house historical vehicle/untreated control values and were, therefore considered of no biological relevance. Under the study conditions, the read across substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation (Envigo, 2017). Based on results of the read across study, similar absence of mutagenicity is expected for the test substance, Cocodimonium hydroxypropyl hydrolysed wool.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study was conducted to determine the genotoxic potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed silk' (active: 51%), using Ames test method, according to OECD Guideline 471, EU Method B13/14 and the USA, EPA OCSPP harmonized guideline, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the read across substance using both the Ames plate incorporation (Experiment 1) and pre-incubation (Experiment 2) methods at eight dose levels (i.e., 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation method) was predetermined and ranged from 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh read across substance formulations. The dose range was amended for Experiment 2, following the results of Experiment 1 and ranged from 0.15 to 500 µg/plate . There were eight read across substance concentrations(i.e., 0.15, 0.5, 1.5, 5, 15, 50, 150 500 μg/plate),which were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the toxic limit of the read across substance following the change in test methodology. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the first mutation test (Experiment 1), the read across substance induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of metabolic activation (S9‑mix) initially from 150 µg/plate. Consequently, the toxic limit of the test substance was selected as the maximum dose level in the second mutation test (Experiment 2). Results from the second mutation test showed that the read across substance induced a stronger toxic response employing the pre‑incubation modification with weakened bacterial background lawns initially noted in the absence of S9‑mix from 50 µg/plate (all Salmonella strains) and 150 µg/plate (WP2uvrA). In the presence S9 -mix, weakened bacterial background lawns were initially noted from 150 µg/plate (TA100 and TA1535) and 500 µg/plate (TA98, TA1537 and WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the read across substance varied slightly between strain type, exposures with or without S9 -mix and experimental methodology. No read across substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the read across substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the read across substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method).Minor statistical values were noted in Experiment 2 (TA100 at 0.5 and 15 µg/plate in the absence of S9 -mix), however these responses were within the in-house historical vehicle/untreated control values and were, therefore considered of no biological relevance.Under the study conditions, theread across substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation (Envigo, 2017). Based on results of the read across study, similar absence of mutagenicity is expected for the test substance,Cocodimonium hydroxypropyl hydrolysed wool.

Further, the hydrolysed proteins differences across the read across substances is not expected to have an impact, as the proteins in general are not mutagenic and form and play an important part in the living organisms (such as enzymes, antibodies, hormones, transport or structural proteins, essential elements of the motile and contractile systems) (Lehninger, 1983).

Reference:

1) Principles of biochemistry by Albert L Lehninger. pp 1011. Worth Publishers, New York, January, 1983.

Justification for classification or non-classification

Based on the absence of mutagenic response in the read across in vitro studies, the test substance, 'Cocodimonium hydroxypropyl hydrolysed wool' does not warrant classification for genotoxicity, according to the EU CLP (Regulation 1272/2008/EC).