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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The in vitro gene mutation study in bacteria was conducted on the read-across substance Gas oil (polymer derived) thermal-cracked, full range according to OECD Testing Guideline 471. The test item produced a positive result with metabolic activation. The in vitro gene mutation study in mammalian cells was conducted on the read-across substance Distillates (petroleum), light catalytic cracked according to OECD Testing Guideline 476. The test item produced a positive result with metabolic activation. The cytogenicity study was conducted on the read-across substance Distillates (petroleum), light catalytic cracked according to a method similar to OECD Testing Guideline 475. The test item was considered to be non-mutagenic in vivo under the conditions of the test.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 471. The study was conducted on Gas oil (polymer-derived), thermal cracked, full range, from which the registered substance is derived via steam stripping, and which is compositionally similar to the registered substance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item were assayed in triplicate against each tester strain : 1.5 µg/plate, 5 µg/plate, 15 µg/plate, 50 µg/plate, 150 µg/plate, 500 µg/plate, 1500 µg/plate, and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks. Acetone was therefore selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
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: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48h
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT: histidine (TA98, TA100, TA1535, TA1537) and tryptophan (WP2)

DETERMINATION OF CYTOTOXICITY
- Method: other: scoring of colonies

OTHER:
- Temperature during incubation: 37°C +/- 3°C
Evaluation criteria:
Method : Scoring of revertant colonies.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

The test item caused a visible reduction in the growth of the bacterial background lawns of all the test strains, both with and without metabolic activation at 5000 µg/plate. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate was noted at 5000 µg/plate, but did not prevent the scoring of revertant colonies.

In both Experiments 1 and 2, the test item induced a dose-related, reproductible and statistically significant increase in the frequency of TA98 revertant colonies at and above 15 µg/plate (up to the toxic limit at 5000 µg/plate) in the presence of S9-mix only. At the upper test item dose levels (excluding the maximum dose of 5000 µg/plate) the increases achieved a two-fold increase over the concurrent vehicle controls in both experiments.
Smaller increases were also observed for TA100 at 150 µg/plate in Experiment 1, and at 150 µg/plate and 500 µg/plate in Experiment 2 in the presence of S9-mix only.
There was also a significant increase in TA1535 in the absence of S9-mix at 15, 50 and 150 µg/plate in Experiment 1 only. However, the second experiment did not produce a response at any dose level so, and as a consequence, a third confirmatory experiment was performed to obtain consistent results. The third experiment confirmed the result noted in Experiment 2 with no statistically significant increases observed at any test item dose level. Therefore, the result observed in Experiment 1 was considered spurious because it was non-reproductible and the response were noted at low test item concentrations.
No significant increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Spontaneous Mutation Rates (Concurrent Negative Controls)
Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
Experiment 1
100 (100) 25 (28) 15 (21) 27 (30) 20 (15)
98 29 24 36 11
102 29 25 27 15
Experiment 2
80 (84) 13 (13) 23 (17) 16 (22) 9 (9)
98 15 8 31 7
75 12 19 19 11
Experiment 3
    17 (17)            
  17      
  17      

Test Results - Experiment 1 - With Metabolic Activation
  Dose level per plate Number of revertants (mean number of colonies per plate) +/- SD
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
S9-Mix (+) Solvent control (acetone) 68 (110)
36.1
12 (14)
2.1
33 (35)
4.7
25 (28)
3.1
21 (14)
6.1
131 16 31 31 9
130 13 40 27 13
1.5 µg 127 (119)
11.4
13 (12)
1.2
16 (19)
4.6
20 (19)
1.7
8 (12)
6.1
106 11 24 17 19
124 11 16 20 9
5 µg 104 (111)
13.3
13 (14)
5.0
35 (26)
7.8
31 (31)
4.0
15 (10)
4.4
102 19 20 35 8
126 9 24 27 7
15 µg 136 (116)
17.4
9 (14)
6.2
32 (28)
6.1
27 (28)
0.6
11 (10)
5.6
108 17 31 28 4
104 15 21 28 15
50 µg 110 (129)
17.0
15 (17)
2.5
31 (29)
1.5
33 (33)
6.5
15 (18)
2.6
142 17 29 27 20
136 20 28 40 19
150 µg 139 (146)
6.4
13 (14)
5.6
36 (28)
8.5
41 (39)
2.9
9 (14)
5.0
150 9 19 36 19
150 20 29 41 13
500 µg 140 (137)
2.6
11 (11)
0.6
25 (27)
1.5
60 (57)
3.5
19 (18)
2.1
135 12 27 57 20
136 11 28 53 16
1500 µg 136 (133)
5.2
11 (14)
4.2
20 (24)
6.9
55 (57)
2.1
13 (19)
5.1
136 13 20 59 20
127 19 32 58 23
5000 µg 128 (130)
7.2
8 (8)
4.0
36 (29)
8.3
57 (52)
4.6
16 (15)
2.1
124 4 20 49 17
138 12 32 49 13
Positive controls S9-Mix (+) Name 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene benzo(a)pyrene 2-aminoanthracene
Dose level per plate 1 µg 2 µg 10 µg 5 µg 2 µg
N° of revertants 1088 (1204)
321.7
150 (236)
74.5
216 (192)
21.5
111 (141)
35.4
235 (182)
70.3
957 281 174 180 208
1568 277 187 132 102
 
Test Results - Experiment 1 - Without Metabolic Activation
  Dose level per plate Number of revertants (mean number of colonies per plate) +/- SD
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
S9-Mix (-) Solvent control (acetone) 114 (113)
7.0
20 (23)
2.5
15 (17)
6.2
17 (17)
6.5
16 (15)
1.5
120 25 12 11 13
106 23 24 24 15
1.5 µg 116 (112)
3.2
8 (20)
10.8
32 (24)
7.0
11 (13)
5.3
12 (11)
4.0
110 23 21 9 7
11 29 19 19 15
5 µg 94 (106)
10.4
23 (22)
1.7
16 (17)
4.0
8 (13)
54.6
13 (13)
1.5
112 20 13 16 15
112 23 21 16 12
15 µg 100 (105)
9.0
110 (95)
30.4
21 (27)
8.1
19 (19)
6.0
12 (15)
4.9
99 115 36 13 21
115 60 23 25 13
50 µg 92 (105)
11.0
71 (56)
17.9
25 (18)
8.7
25 (16)
7.5
23 (20)
3.1
111 60 20 12 21
11 36 8 12 17
150 µg 120 (112)
24.6
41 (55)
18.5
23 (21)
1.5
24 (22)
4.4
16 (14)
1.7
84 48 21 17 13
131 76 20 25 13
500 µg 118 (117)
13.0
39 (42)
5.8
11 (21)
10.0
24 (21)
3.5
24 (16)
7.2
130 49 31 17 11
104 39 20 21 12
1500 µg 120 (115)
5.0
40 (45)
9.0
24 (22)
4.9
23 (20)
6.1
13 (9)
4.0
110 39 25 13 9
114 55 16 24 5
5000 µg 100 (109)
9.0
17 (24)
7.5
25 (21)
5.3
8 (13)
5.6
13 (8)
4.6
110 32 23 19 4
118 23 15 12 7
Positive controls S9-Mix (-) Name N-ethyl-N-nitro-N-nitrosoguanidine N-ethyl-N-nitro-N-nitrosoguanidine N-ethyl-N-nitro-N-nitrosoguanidine 4-nitroquinoline-N-oxide 9-aminoacridine
Dose level per plate 3 µg 5 µg 2 µg 0.2 µg 80 µg
N° of revertants 524 (565)
121.8
512 (828)
282.8
524 (462)
90.0
269 (295)
36.7
559 (519)
99.6
469 1058 504 337 406
702 913 359 279 593
 
Test Results - Experiment 2 - With Metabolic Activation
  Dose level per plate Number of revertants (mean number of colonies per plate) +/- SD
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
S9-Mix (+) Solvent control (acetone) 116 (91)
22.9
9 (11)
1.5
20 (22)
4.0
15 (18)
8.5
  (8)
3.0
71 12 27 12  
86 11 20 28  
1.5 µg 115 (113)
4.0
9 (8)
0.6
13 (19)
6.0
17 (20)
3.1
  (5)
2.6
115 8 25 19  
108 8 19 23  
5 µg 108 (109)
2.1
9 (9)
0.6
20 (20)
0.6
19 (20)
4.6
  (6)
1.2
111 9 20 25  
107 8 19 16  
15 µg 104 (113)
8.3
9 (9)
0.6
32 (25)
6.5
29 (25)
3.2
  (5)
1.5
120 8 19 24  
116 9 25 23  
50 µg 96 (105)
8.2
11 (12)
4.2
24 (22)
4.0
40 (38)
7.8
  (5)
1.5
107 9 17 29  
112 17 24 44  
150 µg 120 (126)
5.1
8 (10)
4.0
27 (27)
0.0
60 (48)
10.6
  (7)
2.1
130 8 27 44  
127 15 27 40  
500 µg 121 (120)
1.2
13 (11)
2.5
27 (24)
8.3
59 (57)
4.9
8 (10)
4.0
119 8 15 51 8
121 11 31 60 15
1500 µg 88 (91)
16.2
5 (7)
2.1
20 (25)
6.8
52 (52)
0.6
19 (16)
3.1
108 9 33 52 15
76 8 23 53 13
5000 µg 68 (93)
22.0
8 (17)
1.7
31 (30)
5.0
55 (64)
8.5
15 (11)
3.8
104 5 25 72 8
108 8 35 65 9
Positive controls S9-Mix (+) Name 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene benzo(a)pyrene 2-aminoanthracene
Dose level per plate 1 µg 2 µg 10 µg 5 µg 2 µg
N° of revertants 1637 (155_)
131.9
230 (230)
18.5
164 (179)
12.7
222 (222)
15.0
289 (299)
8.7
1406 212 186 237 303
1632 249 186 207 305
 
Test Results - Experiment 2 - Without Metabolic Activation
  Dose level per plate Number of revertants (mean number of colonies per plate) +/- SD
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
S9-Mix (-) Solvent control (acetone) 86 (85)
4.2
12 (14)
4.0
27 (19)
7.2
12 (14)
2.1
7 (8)
2.3
88 12 17 13 7
80 19 13 16 11
1.5 µg 75 (72)
3.0
19 (12)
5.8
13 (15)
2.1
9 (11)
1.5
5 (5)
0.0
72 9 16 11 5
69 9 17 12 5
5 µg 74 (86)
11.1
11 (12)
1.2
16 (17)
2.3
11 (14)
3.1
3 (7)
4.0
96 13 16 15 11
88 11 20 17 7
15 µg 86 (87)
5.0
8 (8)
0.0
20 (16)
4.0
11 (13)
3.2
9 (6)
3.1
92 8 16 17 5
82 8 12 12 3
50 µg 69 (71)
4.4
11 (14)
4.6
21 (20)
2.3
12 (17)
8.7
7 (8)
3.1
68 11 21 12 5
76 19 17 27 11
150 µg 96 (82)
12.5
15 (15)
5.5
17 (22)
4.4
20 (19)
1.7
8 (6)
1.7
72 20 25 20 5
78 9 24 17 5
500 µg 78 (75)
2.6
12 (12)
0.0
17 (20)
3.1
9 (12)
3.1
5 (5)
1.5
73 12 19 11 4
74 12 23 15 7
1500 µg 78 (69)
7.6
12 (12)
0.6
17 (17)
0.0
8 (11)
3.8
5 (4)
1.0
66 12 17 15 4
64 11 17 9 3
5000 µg 63 (68)
4.6
8 (8)
0.6
19 (20)
0.1
17 (13)
4.0
1 (4)
2.3
71 9 20 13 5
71 8 21 9 5
Positive controls S9-Mix (-) Name N-ethyl-N-nitro-N-nitrosoguanidine N-ethyl-N-nitro-N-nitrosoguanidine N-ethyl-N-nitro-N-nitrosoguanidine 4-nitroquinoline-N-oxide 9-aminoacridine
Dose level per plate 3 µg 5 µg 2 µg 0.2 µg 80 µg
N° of revertants 480 (459)
64.7
175 (161)
45.6
441 (440)
18.0
227 (234)
5.8
482 (688)
255.7
386 198 457 237 607
510 110 421 237 974
 
Test Results - Experiment 3 - Without Metabolic Activation
  Dose level per plate Number of revertants (mean number of colonies per plate) +/- SD
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
S9-Mix (-) Solvent control (acetone)     21 (16)
4.2
           
  13      
  15      
1.5 µg     23 (18)
4.2
           
  17      
  15      
5 µg     17 (12)
4.6
           
  9      
  9      
15 µg     17 (15)
2.5
           
  12      
  15      
50 µg     9 (14)
4.2
           
  17      
  15      
150 µg     8 (16)
6.9
           
  20      
  20      
500 µg     28 (16)
11.4
           
  7      
  13      
1500 µg     9 (10)
5.0
           
  5      
  15      
5000 µg     8 (13)
6.2
           
  20    
  11      
Positive controls S9-Mix (-) Name N-ethyl-N-nitro-N-nitrosoguanidine N-ethyl-N-nitro-N-nitrosoguanidine N-ethyl-N-nitro-N-nitrosoguanidine 4-nitroquinoline-N-oxide 9-aminoacridine
Dose level per plate 3 µg 5 µg 2 µg 0.2 µg 80 µg
N° of revertants     191 (173)
16.6
           
  171      
  158      
Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation

The test substance was found to be genotoxic in vitro, in the presence of metabolic activation only.
Executive summary:

The in vitro genotoxicity in bacteria on the substance was determined in accordance with the OECD Guideline for Testing of Chemicals 471. Ames plate incorporation (Experiment 1) and pre-incubation (Experiment 2).

The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate.

The test item caused a visible reduction in the growth of the bacterial background lawns of all the test strains, both with and without metabolic activation at 5000 µg/plate. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate was noted at 5000 µg/plate, but did not prevent the scoring of revertant colonies.

In both Experiments 1 and 2, the test item induced a dose-related, reproductible and statistically significant increase in the frequency of TA98 revertant colonies at and above 15 µg/plate (up to the toxic limit at 5000 µg/plate) in the presence of S9-mix only. At the upper test item dose levels (excluding the maximum dose of 5000 µg/plate) the increases achieved a two-fold increase over the concurrent vehicle controls in both experiments.

Smaller increases were also observed for TA100 at 150 µg/plate in Experiment 1, and at 150 µg/plate and 500 µg/plate in Experiment 2 in the presence of S9-mix only.

There was also a significant increase in TA1535 in the absence of S9-mix at 15, 50 and 150 µg/plate in Experiment 1 only. However, the second experiment did not produce a response at any dose level so, and as a consequence, a third confirmatory experiment was performed to obtain consistent results. The third experiment confirmed the result noted in Experiment 2 with no statistically significant increases observed at any test item dose level. Therefore, the result observed in Experiment 1 was considered spurious because it was non-reproductible and the response were noted at low test item concentrations.

No significant increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains.

It was therefore concluded that the test item was mutagenic with metabolic activation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

The in vitro genotoxicity in bacteria on the read-across substance Gas oil (polymer derived) thermal-cracked, full range was determined in accordance with the OECD Guideline for Testing of Chemicals 471. Ames plate incorporation (Experiment 1) and pre-incubation (Experiment 2).

The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate.

The test item caused a visible reduction in the growth of the bacterial background lawns of all the test strains, both with and without metabolic activation at 5000 µg/plate. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate was noted at 5000 µg/plate, but did not prevent the scoring of revertant colonies.

In both Experiments 1 and 2, the test item induced a dose-related, reproductible and statistically significant increase in the frequency of TA98 revertant colonies at and above 15 µg/plate (up to the toxic limit at 5000 µg/plate) in the presence of S9-mix only. At the upper test item dose levels (excluding the maximum dose of 5000 µg/plate) the increases achieved a two-fold increase over the concurrent vehicle controls in both experiments.

Smaller increases were also observed for TA100 at 150 µg/plate in Experiment 1, and at 150 µg/plate and 500 µg/plate in Experiment 2 in the presence of S9-mix only.

There was also a significant increase in TA1535 in the absence of S9-mix at 15, 50 and 150 µg/plate in Experiment 1 only. However, the second experiment did not produce a response at any dose level so, and as a consequence, a third confirmatory experiment was performed to obtain consistent results. The third experiment confirmed the result noted in Experiment 2 with no statistically significant increases observed at any test item dose level. Therefore, the result observed in Experiment 1 was considered spurious because it was non-reproductible and the response were noted at low test item concentrations.

No significant increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains.

It was therefore concluded that the result was positive with metabolic activation.

The in vitro genotoxicity in bacteria of the read-across substance Distillates (petroleum), light catalytic cracked was determined by a GLP accredited laboratory using a method similar to OECD 476.

In the mouse lymphoma forward mutation assay, in vitro treatments of the mouse lymphoma cell line L5178Y, with the test substance induced significant increases in the mutation frequency at the thymidine kinase (TK) locus only in the presence of metabolic activation. The cells were exposed to the test substance for four hours in the presence and absence of rat liver S9 metabolic activation and the test material was soluble up to 50nl/ml. Under nonactivation conditions, the test substance was assayed at an applied concentration range of 5nl/ml to 60nl/ml without inducing significant increases in the mutant frequency. Non detectable to moderate toxicities were induced and a small increase in concentration from 60nl/ml to 80nl/ml was excessively toxic and the treatment could not be cloned. In the presence of the metabolic activation mix, the test substance was assayed from 2.5 nl/ml to 25 nl/ml and significant dose-dependant increases in the mutant frequency were induced above 5.0nl/ml where low to very high toxicities were induced.

It was therefore concluded that the result was positive with metabolic activation.

The in vivo cytogenicity of the read-across substance Distillates (petroleum), light catalytic cracked was determined according to a method similar to the OECD Guideline for Testing of chemicals 475. Chromosome aberrations were evaluated in Sprague-Dawley rats after administration by intraperitoneal route.

The results were negative for acute cytogenotoxicity. The animals were exposed to 0.2, 0.67, or 2g/kg of the test item using corn oil as a vehicle for 6, 24 and 48 hours before the rat was sacrificed and the bone marrow removed. The incidence of cells with aberrations was not significantly increased when compared to the vehicle control. Triethylenemelamine was used as a positive control. Rats showed signs of lethargic behaviour, scruffy coat, blood around nose, disorientation and uncoordination, and death on a dose and time related correlation.

The test item was therefore evaluated as none genotoxic under the conditions of this assay.

Evaluation of genetic toxicity is addressing two different mechanisms: gene mutation and action on chromosomes (aneugenicity and clastogenicity). The in vitro gene mutation assay in bacteria is used as a screening test as it is a well-known method able to identify a large amount of genotoxic substances; however, as it is performed on bacteria, it can identify issues that would not happen in mammalian cells, justifying the need to perform further testing.

The two in vitro assays in mammalian cells required in Annex VIII of REACH are designed in order to assess respectively the potential of the substance to induce gene mutation and to affect chromosomes. Depending on the outcome of these in vitro assays on mammalian cells, in vivo assays can be proposed to fully investigate the issues previously identified. Therefore, the in vivo studies must be selected depending on these results.

For the registered substance, read-across substances considered as representing worse-case scenarios for genetic toxicity were selected. Regarding the potential to affect chromosomes, only in vivo studies were available, which came back as negative. Regarding the potential to induce gene mutation, only in vitro studies were available, which came back as negative without metabolic activation and positive with metabolic activation. From these results, the registered substance is not considered as affecting chromosomes, but it can be expected as inducing gene mutation, which could be confirmed by an in vivo study selected to address this particular mechanism, in accordance with Annex VIII of REACH. However, it is not considered as necessary to perform an additional animal study as it has been decided to classify the registered substance as Mut 1B based on these positive in vitro results.


Justification for selection of genetic toxicity endpoint
Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 471. The study was conducted on Gas oil (polymer-derived), thermal cracked, full range, from which the registered substance is derived via steam stripping, and which is compositionally similar to the registered substance.

Justification for classification or non-classification

As the in vivo cytogenicity study on a read-across substance provided a negative result under the conditions of the test, in accordance with the Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures, the registered substance does not meet the criteria for classification as mutagenic.

However, the positive results obtained on read-across substances during the in vitro gene mutation studies, in bacteria and mammalian, are suggesting that the genotoxicity of the registered substance would be associated with a gene mutation activity. The in vivo cytogenicity study is not considered as sufficient to assess if the registered substance does display a gene mutation activity in vivo. Therefore, it is proposed a classification of the registered substance as Mut 1B, in order to provide a conservative approach.