Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro - bacteria - Ames test , result: positive in Salmonella typhimurium strains TA1535 and TA100 with and without metabolic activation and in TA102 with metabolic activation

Read-across: Genetic toxicity in vitro - mammalian cells mouse lymphoma L5178Y cells, result: negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-12-19 - 2002-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
other: USA, EPA (TSCA) OPPTS harmonised guidelines
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium LT2
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
10% liver S9-mix in standard co-factors
Test concentrations with justification for top dose:
Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 5, 150, 500, 1500 and 5000 µg/plate
Mutation study - Experiment 1: 15, 5, 150, 500, 1500 and 5000 µg/plate
Mutation study - Experiment 1: 1.5, 5, 15, 5, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl formamide
- Justification for choice of solvent/vehicle: Dimethyl formamide is an acceptable vehicle for use in this test system
Untreated negative controls:
yes
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
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION: Exposure duration: 48 hours
NUMBER OF REPLICATIONS: in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

TESTER STRAINS:
The strains used in this assay were all mutants derived from Salmonella typhimurium LT2 and were those recommended for general screening.
TA100
TA 1535 sensitive to agents inducing base-pair substitution
TA102 sensitive to agents inducing frame-shift mutations
TA98
The strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 and were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa, R factors, uvrB mutation and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 250427 06/06) and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.


PROCEDURE
Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100), 2 ml of molten, trace histidine supplemented, top agar, 0.1 ml of test material formulation, 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel- Bonner Minimal agar (30 ml/plate). Ten doses of the test material and a vehicle control (dimethyl formamide) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Study - Experiment 1
Six concentrations of the test material (15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation Study - Experiment 2
The second experiment was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was amended following observations made from Experiment 1 and was as follows:
TA100 and TA1535 (with and without S9), TA98 (with S9 only): 1.5, 5, 15, 50, 150, 500, 1500 ug/plate
TA102 (with and without S9): 5, 15, 50, 150, 500, 1500, 5000 ug/plate
TA98 (without S9 only), TA1537 (with and without S9): 15, 50,150, 500,1500 ug/plate
Additional dose levels were included to allow for the toxicity of the test material, ensuring there were a minimum of four non-toxic doses and, in the case of TA100 and TA1535, to determine the level at which the test material first induced mutations.

ACCEPTANCE CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkMlOl plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels. There should be no evidence of excessive contamination.

















Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression(5)) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
not specified
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
15 - 500 µg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
15 - 150 µg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
10 - 500 µg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The test material was toxic initially at and above 1500 ug/plate to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be effectively sterile

Preliminary study

The number of revertant colonies for the toxicity assay were:

With (+) or without (-) Metabolic Activation Strain Dose (µg/plate)
0 0,15 0,5 1,5 5 15 50 150 500 1500 5000
- TA 100 94 73 72 93 112 160 231 257 310 0V 0V
+ TA100 148 104 93 126 147 283 322 300 255 249 0T
T = toxic, no bacterial background lawn
V = very weak bacterial background lawn

Mutation Study

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Study.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 to Table 5.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies in all of the tester strains, both with and without S9-mix, at and above 1500 µg/plate. The test material was, therefore, tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial tester strain type and presence or absence of S9-mix. A black, fine, powdery precipitate was observed at and above 5000 µg/plate, this did not prevent the scoring of revertant colonies.

Dose-related, reproducible and statistically significant increases were observed at sub-toxic dose levels of the test material. TA100 and TA1535 exhibited statistically significant increases from 15 to 500 µg/plate both with and without S9-mix. TA102 exhibited reproducible increases in the presence of S9 only from 15 to 150 µg/plate.

No reproducible increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains, with any dose of the test material, either with or without metabolic activation.

Judging by the type of Salmonella strains exhibiting dose-related increases (TA100, TA1535 and TA102), the test material was concluded to be a base-pair mutagen.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1: Spontaneous Mutation Rates (Concurrent Negative Controls)
Experiment 1
Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 TA102 TA98 TA1537
94 (97) 27 (23) 318 (322) 26 (23) 6 (13)
82 21 325 23 18
116 22 322 20 16
                
Experiment 2
Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 TA102 TA98 TA1537
112 (101) 26 (22) 302 (308) 26 (21) 13 (12)
94 16 303 24 9
96 24 318 13 15

Table 2: Test Results: Experiment 1 - Without Metabolic Activation
Test Period From: 04 January 2002 To: 07 January 2002
With or without S9-Mix Test substance concentration (µg/plate) Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 TA102 TA98 TA1537
- 0 87
81
76
(81)
5.5#
32
28
26
(29)
3.1
313
341
328
(327)
14.0
24
26
19
(23)
3.6
14
14
8
(12)
3.5
- 15 222
210
270
$$$
(234)
31.7
34
46
50
$$$
(43)
8.3
309
329
335
(324)
13.6
36
24
25
(28)
6.7
11
9
12
(11)
1.5
- 50 380
352
360
$$$
(364)
14.4
57
65
75
$$$
(66)
9,0
329
330
365
(341)
20.5
28
27
32
(29)
2.6
14
12
16
(14)
2,0
- 150 280
336
291
$$$
(302)
29.7
78
73
66
$$$
(72)
6,0
305
344
368
(339)
31.8
23
31
44
(33)
10.6
11
19
22
(17)
5.7
- 500 252
244
248
$$$
(248)
4.0
64
54
61
$$$
(60)
5.1
281
250
274
(268)
16.3
28
27
31
(29)
2.1
16
15
13
(15)
1.5
- 1500 0V
0V
0V
(0)
0.0
0V
0V
0V
(0)
0.0
94
104
92
(97)
6.4
0V
0V
0V
(0)
0.0
0T
0T
0T
(0)
0.0
- 5000 0TP
0TP
0TP
(0)
0.0
0TP
0TP
0TP
(0)
0.0
0TP
0TP
0TP
(0)
0.0
0TP
0TP
0TP
(0)
0.0
0TP
0TP
0TP
(0)
0.0
Positive controls

S9-Mix

-
Name ENNG ENNG MMC 4NQO 9AA
Concentration (µg/plate) 3 5 0.5 0.2 80
No. Colonies per plate 548
513
438
(500)
56.2
453
311
289
(351)
89.0
809
919
924
(884)
65.0
110
117
112
(113)
3.6
1766
1924
2215
(1968)
227.8
ENNG
4NQO
9AA
MMC
P
T
V
$$$
#
N-ethyl-N'-nitro-N-nitrosoguanidine
4-Nitroquinoline-1-oxide
9-Aminoacridine
Mitomycin C
Precipiate
Toxic, no bacterial background lawn
Very weak bacterial background lawn
p < = 0.005
Standard deviation

Table 3: Test results: Experiment 1 - With metabolic activation
Test Period From: 04 January 2002 To: 07 January 2002

With or

without S9-Mix

Test substance concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 TA102 TA98 TA1537
+ 0 84
75
95
(85)
10.0#
18
15
8
(14)
5.1
324
381
361
(327)
14.0
29
40
34
(34)
5.5
18
19
17
(18)
1.0
+ 15 196
188
200
$$$
(195)
6.1
52
37
50
$$$
(46)
8.1
491
474
479
(324)
13.6
53
44
59
$$$
(52)
7.5
26
15
21
(21)
5.5
+ 50 312
220
236
$$$
(256)
49.2
53
41
54
$$$
(49)
7.2
483
454
478
(341)
20.5
50
50
48
**
(49)
1.2
16
28
22
(22)
6.0
+ 150 320
316
272
$$$
(303)
26.6
66
55
35
$$$
(52)
15.7
483
457
453
(339)
31.8
50
46
50
**
(49)
2.3
19
27
22
(23)
4.0
+ 500 276
263
274
$$$
(271)
7.0
43
46
46
$$$
(45)
1.7
346
319
326
(268)
16.3
23
34
40
(32)
8.6
13
15
25
(18)
6.4
+ 1500 0T
0T
0T
(0)
0.0
0T
0T
0T
(0)
0.0
155
136
142
(97)
6.4
0V
0V
0V
(0)
0.0
0T
0T
0T
(0)
0.0
+ 5000 0TP
0TP
0TP
(0)
0.0
0TP
0TP
0TP
(0)
0.0
24P
28P
24P
(0)
0.0
0TP
0TP
0TP
(0)
0.0
0TP
0TP
0TP
(0)
0.0

Positive

controls

S9-Mix

+

Name 2AA 2AA DAN BP 2AA

Concentration

(µg/plate)

1 2 10 5 2

No. Colonies

per plate

1469
1309
1638
(1472)
164.5
177
185
183
(182)
4.2
967
588
600
(718)
215.4
257
264
165
(229)
55.2
446
469
418
(444)
25.5
BP
2AA
DAN
P
T
V
**
$$$
#
Benzo(a)pyrene
2-Aminoanthracene
1,8-Dihydroxyanthraquinone
Precipiate
Toxic, no bacterial bachground lawn
Very weak bacterial background lawn
p < = 0.01
p < = 0.005
Standard deviation

Table 4: Test results: Experiment 2 - Without metabolic activation
Test Period From: 11 January 2002 To: 14 January 2002

With or

without S9-Mix

Test substance

concentration (µg/plate)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 TA102 TA98 TA1537
- 0 86
76
83
(82)
5.1#
15
21
17
(18)
3.1
300
372
348
(340)
36.7
14
17
20
(17)
3.0
6
6
15
(9)
5.2
- 1.5 76
70
68
(71)
4.2
13
18
16
(16)
2.5
N/T N/T N/T
- 5 92
79
93
(88)
7.8
14
18
9
(14)
4.5
343
320
341
(335)
12.7
N/T N/T
- 15 123
129
123
$$$
(125)
3.5
29
22
24
(25)
3.6
365
360
369
(365)
4.5
28
22
25
(25)
3.0
11
9
12
(11)
1.5
- 50 188
196
197
$$$
(194)
4.9
36
28
32
$$$
(32)
4.0
374
377
386
*
(379)
6.2
41
29
33
**
(34)
6.1
12
11
17
(13)
3.2
- 150 268
282
264
$$$
(271)
9.5
37
57
53
$$$
(49)
10.6
400
357
363
(373)
23.3
24
23
39
(29)
9.0
13
14
7
(11)
3.8
- 500 304
267
261
$$$
(277)
23.3
53
42
47
$$$
(47)
5.5
285
298
298
(294)
7.5
22
33
14
(23)
9.5
11
13
12
(12)
1.0
- 1500 0V
0V
0V
(0)
0.0
0V
0V
0V
(0)
0.0
129
C
137
(133)
5.7
0V
0V
0V
(0)
0.0
0V
0V
0V
(0)
0.0
- 5000 N/T N/T 0TP
0TP
0TP
(0)
0.0
N/T N/T

Positive

controls

S9-Mix

-

Name ENNG ENNG MMC 4NQO 9AA

Concentration

(µg/plate)

3 5 0.5 0.2 80

No. Colonies

per plate

473
411
438
(441)
31.1
255
255
244
(251)
6.4
852
801
912
(855)
55.6
114
104
106
(108)
173.5
2429
2132
2125
(2229)
173.5
ENNG
4NQO
9AA
MMC
C
N/T
P
T
V
*
**
$$$
#
N-ethyl-N'-nitro-N-nitrosoguanidine
4-Nitroquinoline-1-oxide
9-Aminoacridine
Mitomycin C
Contaminated
Not tested at this dose level
Precipiate
Toxic, no bacterial bachground lawn
Very weak bacterial background lawn
p < = 0.05
p < = 0.01
p < = 0.005
Standard deviation

Table 5: Test results: Experiment 2 - With metabolic activation
Test Period From: 11 January 2002 To: 14 January 2002
With or without S9-Mix Test substance concentration (µg/plate) Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 TA102 TA98 TA1537
+ 0 80
113
92
(95)
16.7#
12
14
12
(13)
1.2
391
393
370
(385)
12.7
29
44
41
(38)
7.9
18
13
16
(16)
1.5
+ 1,5 83
90
98
(90)
7.5
13
9
12
(11)
2.1
N/T 34
32
27
(31)
3.6
N/T
+ 5 113
106
127
(115)
10.7
19
25
14
*
(19)
5.5
402
434
402
(413)
18.5
32
36
35
(34)
2.1
N/T
+ 15 194
183
202
$$$
(193)
9.5
23
24
33
$$$
(27)
5.5
491
437
468
*
(465)
27.1
41
35
40
(39)
3.2
17
17
20
(18)
1.7
+ 50 303
252
317
$$$
(291)
34.2
38
42
42
$$$
(41)
2.3
543
529
563
$$$
(545)
17.1
38
39
27
(35)
6.7
18
24
26
*
(23)
4.2
+ 150 321
370
310
$$$
(334)
31.9
48
62
47
$$$
(52)
8.4
472
524
515
$$$
(504)
27.8
35
35
40
(37)
2.9
24
17
17
(19)
4.0
+ 500 267
297
305
$$$
(290)
20.0
31
32
36
$$$
(33)
2.6
388
378
275
(347)
62.6
33
32
25
(30)
4.4
13
12
14
(13)
1.0
+ 1500 0V
0V
0V
(0)
0.0
0V
0V
0V
(0)
0.0
196
189
195
(193)
3.8
0V
0V
0V
(0)
0.0
0V
0V
0V
(0)
0.0
+ 5000 N/T N/T 34 SP
43 SP
23 SP
(33)
10.0
N/T N/T
Positive controls

S9-Mix

+
Name 2AA 2AA DAN BP 2AA
Concentration (µg/plate) 1 2 10 5 2
No. Colonies per plate 1728
1747
1746
(1740)
10.7
299
254
286
(280)
23.2
916
904
755
(858)
89.7
165
215
174
(185)
26.7
611
611
636
(619)
14.4
BP
2AA
DAN
P
T
V
*
$$$
#
Benzo(a)pyrene
2-Aminoanthracene
1,8-Dihydroxyanthraquinone
Precipitate
Toxic, no bacterial background lawn
Very weak bacterial background lawn
p < = 0.05
p < = 0.005
Standard deviation
Conclusions:
The test material was considered to be mutagenic under the conditions of this test.
Executive summary:

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines and was conducted in compliance with GLP.

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with suspensions of the test material using the Ames plate incorporation method at up to seven dose levels (0, 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 metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following observations made in Experiment 1 and ranged between 1.5 and 5000 µg/plate depending on bacterial strain type and presence or absence of S9-mix. Additional dose levels were used in both experiments (where applicable) to allow for the toxicity of the test material ensuring there were a minimum of four non-toxic doses.

The vehicle (dimethyl formamide) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies in all of the tester strains, both with and without S9-mix, at and above 1500 µg/plate. The test material was, therefore, tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial tester strain type and presence or absence of S9-mix. A black, fine, powdery precipitate was observed at and above 5000 µg/plate, this did not prevent the scoring of revertant colonies.

Dose-related, reproducible and statistically significant increases were observed at sub-toxic dose levels of the test material. TA100 and TA1535 exhibited statistically significant increases from 15 to 500 µg/plate both with and without S9-mix. TA102 exhibited reproducible increases in the presence of S9 only from 15 to 150 µg/plate.

No reproducible increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains, with any dose of the test material, either with or without metabolic activation.

Judging by the type of Salmonella strains exhibiting dose-related increases (TA100, TA1535 and TA102), the test material was concluded to be a base-pair mutagen.

The test material was considered to be mutagenic under the conditions of this test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Both the target substance Ferbam and the source substance Ziram quickly hydrolyse forming dimethyldithiocabamates. The analogue hypothesis is that both substances dissociate in acidic and neutral aqueous solutions to the common breakdown product dimethyldithiocarbamate, which determines the (eco-)toxicological properties. Therefore, the basis for this analogue approach is the “(Bio)transformation to common compound(s)” (scenario 1). This scenario covers the analogue approach for which the read-across hypothesis is based on (bio) transformation to common compound(s). For the REACH information requirement under consideration, the effects obtained in a study conducted with one source substance are used to predict the effects that would be observed in a study with the target substance if it were to be conducted. The same type of effect(s) or absence of effect is predicted. The predicted strength of the effects may be similar or based on a worst-case approach (Read-Across Assessment Framework, RAAF, ECHA, 2017).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance is Ziram (IUPAC name: zinc bis(dimethyldithiocarbamate , CAS no. 137-30-4; EC no. 205-288-3). Ziram is an organometallic mono-constituent substance with a typical purity of ca. 99% (w/w), ranging from > 98% to 100%. In general, impurities are known and are likely to vary depending on the manufacturing process, but are considered not relevant for classification or labelling of the substance.
The target substance is Ferbam (IUPAC name: N,N-dimethylcarbamodithioate;iron(3+), CAS no. 14484-64-1; EC no. 238-484-2). Also Ferbam is an organometallic mono-constituent substance, with a typical purity of ca. 99% (w/w), ranging from > 98% to 100%. In general, impurities are known and are likely to vary depending on the manufacturing process, but are considered not relevant for classification or labelling of the substance.
The substance identities and all REACH Annex VII and VIII information on physicochemical properties, environmental fate, ecotoxicity and toxicity are summarised in the read-across statement attached under section 13 of this IUCLID file. Both substances are dimethyldithiocarbamates with a central metal. The high structural similarity is also reflected in very similar physicochemical properties. Both substances are primarily used fungicides.

3. ANALOGUE APPROACH JUSTIFICATION
The potential of Ziram to induce mutations was investigated in an in vitro mammalian cell gene mutation test according to OECD TG 476. Ziram was tested in concentrations up to 3 µg/mL in two independent experiments with and without metabolic activation (S9 mix) exposing mouse lymphoma L5178Y cells for 24 hours. No biologically relevant increases in mutant frequency were observed, so that Ziram was negative, i.e. had no mutagenic potential, in this study. Due to the similar hydrolysis, the read-across of this study and the result is justified, as substances are applied in dilution to water.
For further information on the justification of the analogue approach please refer to the read-across statement attached under section 13 of this IUCLID file.

4. DATA MATRIX
Please refer to the data matrix in the read-across statement attached under section 13 of this IUCLID file.
Reason / purpose:
read-across source
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y mouse lymphoma cells (3.7.2C) were obtained from Dr J Cole, University of Sussex. These cells are heterozygous at the thymidine kinase locus, TK+/-. Spontaneous thymidine kinase deficient mutants, TK-/-, were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C. The cells were stored in polypropylene ampoules in heat-inactivated donor horse serum (HiDHS) containing 10% dimethylsulphoxide (DMSO) and used within seven days of recovery from frozen stock. RlOp medium (see below) was used for routine expansion of cultures.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9 cells (S9-mix)
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material was tested for mutagenic potential in the mouse lymphoma thymidine kinase assay. Four independent tests were carried out, two in the absence of exogenous metabolic activation (S-9 mix) and two in the presence of S-9 mix.

Preliminary toxicity test
In the preliminary toxicity test on the test item, treatment with 1.96 - 125 µg/ml in the absence and presence of S-9 mix resulted in Day0 relative survival over this range of 26 - 0% and 51 - 0% respectively compared to the solvent controls. A second preliminary toxicity test was carried out using a continuous treatment period of 24 hours in the absence of S-9 mix. Treatment with 0.01 - 5 µg/ml in the absence of S-9 mix resulted in Day0 relative survival over this range of 113 - 0%. Concentrations used in the main test were based upon these data.
In the absence of S-9 mix treatment of cells with 0.005 - 3 µg/ml in Test 1 using a 3 hour treatment period and 0.0001 - 0.1 µg/ml in Test 2 using a continuous treatment of 24 hours resulted in Day0 relative survivals of 109 - 0% and 109 - 0% respectively. Cultures treated with 0.1, 0.25, 0.5, 1 and 2 µg/ml in Test 1 and 0.001, 0.0025, 0.005, 0.01 and 0.025 µg/ml in Test 2 were plated with and without TFT (the selective agent) to permit measurement of the levels of cloning efficiency and induced mutation. The resulting Day2 cloning efficiencies over this range were 93 - 58% in Test 1 and 86 - 58% in Test 2 relative to the controls.
A statistically significant increase in mutant frequency, which was outside the historical control range, was observed at 2 µg/ml in Test 1. This increase was observed at a Day0 relative survival of 26%. In Test 2, no statistically significant increases in mutant frequency were observed even at levels of extreme toxicity. As no reproducible increases in mutant frequency, which were outside the historical control range, were observed over at least two dose levels, it was therefore concluded that the test material did not demonstrate mutagenic potential in this in vitro mammalian cell mutation assay in the absence of S-9 mix. MMS the positive control, induced highly significant increases in mutant frequency in both tests.
In the presence of S-9 mix treatment of cells with 0.005 - 3 µg/ml in Test 1 and 0.05 - 3 µg/ml in Test 2 respectively resulted in Day0 relative survivals of 118 - 0% and 83 - 4% respectively. Cultures treated with 0.1, 0.25, 0.5, 1 and 2 µg/ml in Test 1 and 1, 1.5, 2,2.5 and 3 µg/ml in Test 2 were plated with and without TFT (the selective agent) to permit measurement of the levels of cloning efficiency and induced mutation. The resulting Day2 cloning efficiencies over this range were 88 - 76% in Test 1 and 97 - 57% in Test 2 relative to the controls.
No statistically significant increases in mutant frequency, which were outside the historical control range, were observed in Test 1 after treatment with the test material. In Test 2, a statistically significant increase which was outside the historical control was observed at 3 µg/ml, but the Day0 relative survival at this level was below the acceptable limit of toxicity (less than 10%). It was therefore concluded that the test item did not demonstrate mutagenic potential in this in vitro mammalian cell mutation assay in the presence of S-9 mix. 20-Methylcholanthrene, the positive control, induced highly significant increases in mutant frequency in both tests.
Conclusions:
It was concluded that the test material did not demonstrate mutagenic potential in this in vitro gene mutation assay and this can also be concluded for ferbam, due to high structural similarities of source and target substance.
Executive summary:

The source test material was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in a subline of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). Two independent tests in the absence of exogenous metabolic activation (S-9 mix) and two independent tests in the presence of S-9 mix were carried out.

Toxicity was observed after treatment with the source test material in all tests in both the absence and the presence of S-9 mix.

No biologically significant increases in mutant frequency were observed in any of the tests, either in the absence or the presence of S-9 mix.

It was concluded that the source test material did not demonstrate mutagenic potential in this in vitro cell mutation assay.

These results can be used to fulfill the information requirements for the chemical substance ferbam due to the high structural similarity of these substances.

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

Genetic toxicity in vivo

Description of key information

Read-across: Genetic toxicity in vivo - in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus, result. negative

Read-across: Genetic toxicity in vivo - in vivo mammalian germ cell study: cytogenicity / chromosome aberration, result: negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Both the target substance Ferbam and the source substance Ziram quickly hydrolyse forming dimethyldithiocabamates. The analogue hypothesis is that both substances dissociate in acidic and neutral aqueous solutions to the common breakdown product dimethyldithiocarbamate, which determines the (eco-)toxicological properties. Therefore, the basis for this analogue approach is the “(Bio)transformation to common compound(s)” (scenario 1). This scenario covers the analogue approach for which the read-across hypothesis is based on (bio) transformation to common compound(s). For the REACH information requirement under consideration, the effects obtained in a study conducted with one source substance are used to predict the effects that would be observed in a study with the target substance if it were to be conducted. The same type of effect(s) or absence of effect is predicted. The predicted strength of the effects may be similar or based on a worst-case approach (Read-Across Assessment Framework, RAAF, ECHA, 2017).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance is Ziram (IUPAC name: zinc bis(dimethyldithiocarbamate , CAS no. 137-30-4; EC no. 205-288-3). Ziram is an organometallic mono-constituent substance with a typical purity of ca. 99% (w/w), ranging from > 98% to 100%. In general, impurities are known and are likely to vary depending on the manufacturing process, but are considered not relevant for classification or labelling of the substance.
The target substance is Ferbam (IUPAC name: N,N-dimethylcarbamodithioate;iron(3+), CAS no. 14484-64-1; EC no. 238-484-2). Also Ferbam is an organometallic mono-constituent substance, with a typical purity of ca. 99% (w/w), ranging from > 98% to 100%. In general, impurities are known and are likely to vary depending on the manufacturing process, but are considered not relevant for classification or labelling of the substance.
The substance identities and all REACH Annex VII and VIII information on physicochemical properties, environmental fate, ecotoxicity and toxicity are summarised in the read-across statement attached under section 13 of this IUCLID file. Both substances are dimethyldithiocarbamates with a central metal. The high structural similarity is also reflected in very similar physicochemical properties. Both substances are primarily used fungicides.

3. ANALOGUE APPROACH JUSTIFICATION
The genotoxicity of Ziram was investigated in a peripheral blood micronucleus test in mice after 89 days of dietary exposure. Groups of 100 mice (50 male and 50 female) received 1, 3, 9 or 27 mg/kg bw Ziram through the diet. In addition, a concurrent negative control group was used, but no positive control. After 89 days of dietary exposure, blood smear were prepared from 10 mice (5 male and 5 female) per group. As Ziram did not reduce the ratio of polychromatic to normochromatic erythrocytes, it was considered as negative, i.e. not causing chromosome damage. The read-across of the study and its results is justified, as both Ziram and Ferbam dissociate in the acidic gastrointestinal environment into the metal cations Zn2+ and Fe3+, respectively, and the common compound dimethyldithiocarbamate (anion), which is subsequently further hydrolysed and absorbed.
For further information on the justification of the analogue approach please refer to the read-across statement attached under section 13 of this IUCLID file.

4. DATA MATRIX
Please refer to the data matrix in the read-across statement attached under section 13 of this IUCLID file.
Reason / purpose:
read-across source
Species:
mouse
Strain:
CD-1
Sex:
male/female
Route of administration:
oral: feed
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
MICRONUCLEATED POLYCHROMATIC ERYTHROCYTE COUNTS (mnp)
The test item did not cause any statistical significant increases in the number of micronucleated polychromatic erythrocytes [P>0.01 using Kruskal-Wallis' test, Jonckheere's test for trend and Spearman's correlation test].

MICRONUCLEATED NORMOCHROMATIC ERYTHROCYTES (mnn)
The test item did not cause any statistically significant increases in the incidence of micronucleated normochromatic erythrocytes [P>0.01 using Kruskal-Wallis' test, Jonckheere's test for trend and Spearman's correlation test].

RATIO OF POLYCHROMATIC TO NORMOCHROMATIC ERYTHROCYTES (pin)
The test item failed to cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes [P>0.01 using Kruskal-Wallis' test, Jonckheere's test for trend and Spearman's correlation test].

Treatment level [ppm]

Ratio p/n (mean) Incidence mnp (mean) Incidence mnn (mean)
Control
0.108 0.9 1.1
25*
0.143 0.6 1.7
75**
0.124 1.2 2.0
225
0.095 0.8 0.7
675 0.135 0.5 1.2

p/n Ratio of polychromatic to normochromatic erythrocytes

mnp No. of micronucleated cells observed per 1000 polychromatic erythrocytes

mnn No. of micronucleated cells observed per 1000 normochromatic erythrocytes

* Prepared at 29 ppm initially to allow for loss during storage

** Prepared at 83 ppm initially to allow for loss during storage

Conclusions:
The source substance did not show any evidence of chromosome-damaging activity or bone marrow cell toxicity after sub-chronic dietary administration in this in vivo test procedure. This can also be concluded for ferbam, due to high structural similarities of source and target substance.
Executive summary:

This report assess the chromosome-damaging activity of the test item following sub-acute dietary administration to mice during an ongoing oncogenicity study (HRC study number ZIR/12) according to EPA Guideline OPP 84 -2 and in accordance with GLP.

Young adult CD-I outbred albino mice were maintained on diet containing the test material at levels of 25, 75, 225 and 675 ppm using the methods described for HRC Study Schedule No. ZIR/12. A group of control mice were maintained on untreated diet.

After 89 days exposure, peripheral blood smears were prepared from 5 male and 5 female animals in each group. The smears were stained using Giemsa then examined by light microscopy for the presence of micronuclei in normochromatic and polychromatic erythrocytes (1000 cells of each type were examined from each animal). The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal.

Mice treated with the test item did not show any significant increase in the frequency of micronucleated normochromatic or polychromatic erythrocytes.

Mice treated with the test item did not show any significant decrease in the ratio of polychromatic to normochromatic erythrocytes (a reduction in this ratio is indicative of bone marrow toxicity).

It is concluded that the test material has not shown any evidence of causing chromosome damage in this in vivo test system.

These results can be used to fulfill the information requirements for the chemical substance ferbam due to the high structural similarity of these substances.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Both the target substance Ferbam and the source substance Ziram quickly hydrolyse forming dimethyldithiocabamates. The analogue hypothesis is that both substances dissociate in acidic and neutral aqueous solutions to the common breakdown product dimethyldithiocarbamate, which determines the (eco-)toxicological properties. Therefore, the basis for this analogue approach is the “(Bio)transformation to common compound(s)” (scenario 1). This scenario covers the analogue approach for which the read-across hypothesis is based on (bio) transformation to common compound(s). For the REACH information requirement under consideration, the effects obtained in a study conducted with one source substance are used to predict the effects that would be observed in a study with the target substance if it were to be conducted. The same type of effect(s) or absence of effect is predicted. The predicted strength of the effects may be similar or based on a worst-case approach (Read-Across Assessment Framework, RAAF, ECHA, 2017).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance is Ziram (IUPAC name: zinc bis(dimethyldithiocarbamate , CAS no. 137-30-4; EC no. 205-288-3). Ziram is an organometallic mono-constituent substance with a typical purity of ca. 99% (w/w), ranging from > 98% to 100%. In general, impurities are known and are likely to vary depending on the manufacturing process, but are considered not relevant for classification or labelling of the substance.
The target substance is Ferbam (IUPAC name: N,N-dimethylcarbamodithioate;iron(3+), CAS no. 14484-64-1; EC no. 238-484-2). Also Ferbam is an organometallic mono-constituent substance, with a typical purity of ca. 99% (w/w), ranging from > 98% to 100%. In general, impurities are known and are likely to vary depending on the manufacturing process, but are considered not relevant for classification or labelling of the substance.
The substance identities and all REACH Annex VII and VIII information on physicochemical properties, environmental fate, ecotoxicity and toxicity are summarised in the read-across statement attached under section 13 of this IUCLID file. Both substances are dimethyldithiocarbamates with a central metal. The high structural similarity is also reflected in very similar physicochemical properties. Both substances are primarily used fungicides.

3. ANALOGUE APPROACH JUSTIFICATION
Ziram’s potential to induce chromosome aberration in spermatogonial mitosis was tested in a mouse germ-cell cytogenetic study according to OECD Test Guideline 483. Five male mice were administered one single dose by oral gavage. Mice were prepared after 6 hours (200 mg/kg bw.), after 24 hours (20, 67 and 200 mg/kg bw) and after 48 hours (200 mg/kg bw.). As the chromosome aberration frequency was not increased at any dose and preparation interval, Ziram was considered to be not mutagenic is this study. The read-across of the study and its results is justified, as both Ziram and Ferbam dissociate in the acidic gastrointestinal environment into the metal cations Zn2+ and Fe3+, respectively, and the common compound dimethyldithiocarbamate (anion), which is subsequently further hydrolysed and absorbed.
For further information on the justification of the analogue approach please refer to the read-across statement attached under section 13 of this IUCLID file.

4. DATA MATRIX
Please refer to the data matrix in the read-across statement attached under section 13 of this IUCLID file.
Reason / purpose:
read-across source
Species:
mouse
Strain:
NMRI
Sex:
male
Route of administration:
oral: feed
Dose / conc.:
200 mg/kg bw (total dose)
Remarks:
preparation after 6 hours after exposure
Dose / conc.:
20 mg/kg bw (total dose)
Remarks:
preparation after 24 hours after exposure
Dose / conc.:
67 mg/kg bw (total dose)
Remarks:
preparation after 24 hours after exposure
Dose / conc.:
200 mg/kg bw (total dose)
Remarks:
preparation after 24 hours after exposure
Dose / conc.:
200 mg/kg bw (total dose)
Remarks:
preparation after 48 hours after exposure
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
pre-experiment
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRE-EXPERIMENT FOR TOXICITY
In a series of pre-experiments males received orally single doses of 100, 200, 300, or 400 mg/kg b.w., respectively. The dose level which was characterized as to be non-lethal was 200 mg/kg b.w.. The treated animals expressed reduction of spontaneous activity.
After administration of this dose scorable spermatogonia preparations were attained.

Summary of Results - Table 3

Table 3: Summary of Results

experimental group dose mg/kg b.w. preparation p. admin, hours number of cells scored % aberrant incl. Gaps cells excl. Gaps mitotic index, %
1 - test article 200 6 500 0.0 0.0 1.40
2 - negative control 0 24 500 0.4 0.2 2.80
3 - test article 20 24 500 0.0 0.0 1.40
4 - test article 67 24 500 0.2 0.0 2.34
5 - test article 200 24 500 0.2 0.2 1.84
6 - positive control 140 24 200 8.0 7.5 0.34
7 - test article 200 48 500 0.8 0.4 1.16
Analysis of aberration types in each 500 cells*
group g ig b ib f if d id ma ex cd
1 0 0 0 0 0 0 0 0 0 0 0
2 0 1 0 0 1 0 0 0 0 0 0
3 0 0 0 0 0 0 0 0 0 0 0
4 0 1 0 0 0 0 0 0 0 0 0
5 0 0 0 0 1 0 0 0 0 0 0
6 3 0 5 0 3 5 0 0 0 2 0
7 2 0 0 0 2 0 0 0 0 0 0
* group 6: 200 cell were scored
g = gab; ig = iso-gab; b = break; ib = iso-break; f = fragment; if = iso-fragment; d = deletion, id= iso-deletion; ma = multiple aberration (more than 5 events, excluding gabs, in one cell, only exchanges, but no other aberrations, were recorded in theses cells); ex = exchange; cd = chromosome disintegration (= pulverization)
Conclusions:
In conclusion, it can be stated that during the mutagenicity assay described and under the experimental conditions reported, the source test article did not induce chromosome aberrations in spermatogonia of the mouse. This can also be concluded for ferbam, due to high structural similarities of source and target substance.
Executive summary:

This GLP in vivo study was performed to investigate the potential of the test item to induce chromosome aberrations in spermatogonial mitoses by means of the mouse germ-cell cytogenetic assay according to EPA OPP 48 -2 and OECD Guideline 483 .

The test article was formulated in 0.5 % carboxymethylcellulose suspension. The volume administered orally was 10 ml/kg body weight (b.w.) at 6 h (24 h and 48 h) after a single application of the test article spermatogonial mitoses were collected for chromosome aberration analysis.

Five animals per test group (positive control only four animals) were evaluated for the occurrence of cytogenetic damage. 100 well spread metaphases per animal of the negative control and of the test article groups and 50 well spread metaphases per animal of the positive control group were scored for gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations.

The test article was tested in this cytogenetic assay in the following dose levels:

6 h preparation interval: 200 mg/kg b.w.

24 h preparation interval: 20, 67, and 200 mg/kg b.w.

48 h preparation interval: 200 mg/kg b.w.

The highest dose administered was determined in pre-experiments for toxicity to be the maximum tolerated dose. All treated animals expressed toxic reactions.

At no preparation interval the chromosome aberration frequency was substancially enhanced as compared to the negative control.

An appropiate reference mutagen was used as positive control and snowed a distinct increase over the spontaneous aberration frequency.

In conclusion, it can be stated that during the mutagenicity assay described and under the experimental conditions reported, treatment with the test article did not result in a significant enhancement of the chromosome aberration frequency in spermatogonia of the mouse at any preparation interval or dose level tested as compared to the negative control value. Therefore, the test item is considered to be non-mutagenic in this chromosome aberration assay.

These results can be used to fulfill the information requirements for the chemical substance ferbam due to the high structural similarity of these substances.

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

Additional information

Genetic toxicity in vitro - bacteria - Ames test

in the key study (Bowles, 2002) the method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines and was conducted in compliance with GLP.

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with suspensions of the test material using the Ames plate incorporation method at up to seven dose levels (0, 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 metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following observations made in Experiment 1 and ranged between 1.5 and 5000 µg/plate depending on bacterial strain type and presence or absence of S9-mix. Additional dose levels were used in both experiments (where applicable) to allow for the toxicity of the test material ensuring there were a minimum of four non-toxic doses.

The vehicle (dimethyl formamide) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies in all of the tester strains, both with and without S9-mix, at and above 1500 µg/plate. The test material was, therefore, tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial tester strain type and presence or absence of S9-mix. A black, fine, powdery precipitate was observed at and above 5000 µg/plate, this did not prevent the scoring of revertant colonies.

Dose-related, reproducible and statistically significant increases were observed at sub-toxic dose levels of the test material. TA100 and TA1535 exhibited statistically significant increases from 15 to 500 µg/plate both with and without S9-mix. TA102 exhibited reproducible increases in the presence of S9 only from 15 to 150 µg/plate.

No reproducible increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains, with any dose of the test material, either with or without metabolic activation.

Judging by the type of Salmonella strains exhibiting dose-related increases (TA100, TA1535 and TA102), the test material was concluded to be a base-pair mutagen.

The test material was considered to be mutagenic under the conditions of this test.

Read-across approach: Genetic toxicity in vitro - mammalian cell mutation assay

The test material was tested for mutagenic potential in the key in vitro mammalian cell mutation assay (Ransom, 1999). This test system is based on detection and quantitation of forward mutation in a subline of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). Two independent tests in the absence of exogenous metabolic activation (S-9 mix) and two independent tests in the presence of S-9 mix were carried out.

Toxicity was observed after treatment with the test material in all tests in both the absence and the presence of S-9 mix.

No biologically significant increases in mutant frequency were observed in any of the tests, either in the absence or the presence of S-9 mix.

It was concluded that the test material did not demonstrate mutagenic potential in this in vitro cell mutation assay.

These results can be used to fulfill the information requirements for the chemical substance ferbam due to the high structural similarity of these substances.

Read-across approach: Genetic toxicity in vivo - mammalian germ cell study: cytogenicity / chromosome aberration

This key report (Proulock, 1992) assess the chromosome-damaging activity of the test item following sub-acute dietary administration to mice during an ongoing oncogenicity study (HRC study number ZIR/12) according to EPA Guideline OPP 84 -2 and in accordance with GLP.

Young adult CD-1 outbred albino mice were maintained on diet containing the test material at levels of 25, 75, 225 and 675 ppm using the methods described for HRC Study Schedule No. ZIR/12. A group of control mice were maintained on untreated diet.

After 89 days exposure, peripheral blood smears were prepared from 5 male and 5 female animals in each group. The smears were stained using Giemsa then examined by light microscopy for the presence of micronuclei in normochromatic and polychromatic erythrocytes (1000 cells of each type were examined from each animal). The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal.

Mice treated with the test item did not show any significant increase in the frequency of micronucleated normochromatic or polychromatic erythrocytes.

Mice treated with the test item did not show any significant decrease in the ratio of polychromatic to normochromatic erythrocytes (a reduction in this ratio is indicative of bone marrow toxicity).

It is concluded that the test material has not shown any evidence of causing chromosome damage in this in vivo test system.

These results can be used to fulfill the information requirements for the chemical substance ferbam due to the high structural similarity of these substances.

Read-across approach: Genetic toxicity in vivo - mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

This GLP in vivo key study (Völkner, 1992) was performed to investigate the potential of the test item to induce chromosome aberrations in spermatogonial mitoses by means of the mouse germ-cell cytogenetic assay according to EPA OPP 48 -2 and OECD Guideline 483.

The test article was formulated in 0.5 % carboxymethylcellulose suspension. The volume administered orally was 10 ml/kg body weight (b.w.) at 6 h (24 h and 48 h) after a single application of the test article spermatogonial mitoses were collected for chromosome aberration analysis.

Five animals per test group (positive control only four animals) were evaluated for the occurrence of cytogenetic damage. 100 well spread metaphases per animal of the negative control and of the test article groups and 50 well spread metaphases per animal of the positive control group were scored for gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations.

The test article was tested in this cytogenetic assay in the following dose levels:

6 h preparation interval: 200 mg/kg b.w.

24 h preparation interval: 20, 67, and 200 mg/kg b.w.

48 h preparation interval: 200 mg/kg b.w.

The highest dose administered was determined in pre-experiments for toxicity to be the maximum tolerated dose. All treated animals expressed toxic reactions.

At no preparation interval the chromosome aberration frequency was substancially enhanced as compared to the negative control.

An appropiate reference mutagen was used as positive control and snowed a distinct increase over the spontaneous aberration frequency.

In conclusion, it can be stated that during the mutagenicity assay described and under the experimental conditions reported, treatment with the test article did not result in a significant enhancement of the chromosome aberration frequency in spermatogonia of the mouse at any preparation interval or dose level tested as compared to the negative control value. Therefore, the test item is considered to be non-mutagenic in this chromosome aberration assay.

These results can be used to fulfill the information requirements for the chemical substance ferbam due to the high structural similarity of these substances.

Overall conclusion

Although the in vitro Ames test with ferbam revealed a mutagenic potential of the substance in three of the bacteria strains, the negative in vitro mammalian cell gene mutation test (MLA) and further negative in vivo (mammalian somatic cell study: cytogenicity / erythrocyte micronucleus and mammalian germ cell study: cytogenicity / chromosome aberration) data available for the ziram read across substance, lead to the conclusion that the substance is neither mutagenic, nor genotoxic, nor induces micronuclei. Therefore the target substance is considered to be not mutagenic nor genotoxic.

Justification for classification or non-classification

Although the in vitro Ames test with ferbam revealed a mutagenic potential of the substance in three of the bacteria strains, the negative in vitro mammalian cell gene mutation test (MLA) and further negative in vivo data (mammalian somatic cell study: cytogenicity / erythrocyte micronucleus and mammalian germ cell study: cytogenicity / chromosome aberration) available for the ziram read across substance, lead to the conclusion that the substance is neither mutagenic, nor genotoxic, nor induces micronuclei. Therefore, the target substance is considered to be not mutagenic nor genotoxic. According to the Regulation (EC) No 1272/2008 the test item ferbam, due to the high structural similarity of these substances, does not meet the criteria for classification and will not require labelling as a mutagen.


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