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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria (similar to OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and E. coli WP2uvrA with and without metabolic activation (RL 1)

In vitro gene mutation study in bacteria (similar to OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and E. coli WP2uvrA with and without metabolic activation (RL 1)

In vitro gene mutation study in bacteria (similar to OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation (RL 1)

In vitro cytogenicity assays in chinese hamster ovary cells (CHO) (similar to OECD 473): positive with and negative without metabolic activation (RL 2)

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:
27 Feb - 15 Mar 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon and trp operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
Pre-experiment: 4, 20, 100, 500, 2500 and 10000 µg/plate with and without metabolic activation
Main Experiment:
Experiment I:
The pre-experiment served as Experiment I.
Experiment II:
4, 20, 100, 500, 2500 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
benzo(a)pyrene
other: -S9: 2-nitrofluorene (5 µg/plate) for TA1538 and TA98; N-Methyl-N'-nitro-N-nitrosoguanidine (2.5 µg/plate) for WP2uvrA; +S9: 2-aminoanthracene (0.5, 1 or 10 µg/plate) for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 2 and 3 replications each in 2 independent experiments (Experiment 1 and 2), respectively.

DETERMINATION OF CYTOTOXICITY
- Method: A reduced rate of spontaneously occuring colonies and visible thinning of the bacterial lawn were used as indicator for toxicity.

OTHER EXAMINATIONS:
Sterility check: A sterility check was performed with agar plates only containing the test substance with and without S9 mix.
Statistics:
Mean values were calculated
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: ≥ 2500 µg/plate without metabolic activation and ≥ 500 µg/plate with metabolic activation; Experiment 2: ≥ 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipiation of the test substance on the plates was observed in the highest concentration of 10000 µg/plate with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment served as Experiment I (all strains were tested in the pre-experiment).

HISTORICAL CONTROL DATA
- Positive historical control data: The historical control data of the positive controls were not provided in the study report, but the control plates with the background control gave the expected number of colonies.
- Negative (solvent/vehicle) historical control data: No historical control data of the vehicle control was provided in the study report, but the control plates with the positive controls gave the expected number of colonies.

OTHER EXAMINATIONS:
Sterility of S-9 Mix and the test compound was indicated by the absence of contamination on the test material and S-9 Mix sterility check plates.

Table 1: Summary of Experiment 1

EXPERIMENT 1
S9-Mix Without
Test item (µg/plate) TA100 TA1535 TA1537 TA1538 TA98 WP2uvrA
0 144 19 10 12 27 16
4 164 18 8 10 19 27
20 161 14 11 13 24 22
100 175 14 8 13 27 22
500 160 11 7 14 23 17
2500 9 (ibl) ibl nbl 0 ibl 0 (ibl) 4 (ibl)
10000 0 P (nbl) 0 P (nbl) 0 P (nbl) 0 P (nbl) 0 P (nbl) 0 P (nbl)
Positive controls SA SA 9-AA 2-NF 2-NF MNNG
Concentrations (ÎĽg/plate) 1 1 50 5 5 2.5
Mean No. of colonies/plate 560 384 95 786 450 221
S9-Mix With
Test item (µg/plate) TA100 TA1535 TA1537 TA1538 TA98 WP2uvrA
0 145 15 12 16 27 25
4 174 13 11 13 31 23
20 185 13 9 18 31 19
100 196 12 15 17 35 25
500 130 (ibl) 7 11 17 30 19
2500 23 (ibl) 2 (ibl) 0 (ibl) 0 (ibl) 1 (ibl) 4
10000 0 P (nbl) 0 P (nbl) 0 P (ibl) 0 P (nbl) 0 P (nbl) 0 P (nbl)
Positive control 2-AA 2-AA 2-AA 2-AA 2-AA 2-AA
Concentrations (ÎĽg/plate) 0.5 1 1 0.5 0.5 10
Mean No. of colonies/plate 680 156 109 538 545 272
Positive control B(a)P B(a)P B(a)P B(a)P B(a)P B(a)P
Concentrations (ÎĽg/plate) 10 10 10 10 10 10
Mean No. of colonies/plate 449 29 163 269 704 57

ibl: incomplete background lawn in all plates

(ibl): incomplete background lawn in individual plates

nbl: no background lawn

(nbl): no background lawn in individual plates

P: precipitation

SA: sodium azide

9-AA: 9 -aminoacridine

2-NF: 2 -nitrofluorene

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine

2-AA: 2 -aminoanthracen

B(a)P: benzo(a)pyrene

Table 2: Summary of Experiment 2

EXPERIMENT 2
S9-Mix Without
Test item (µg/plate) TA100 TA1535 TA1537 TA1538 TA98 WP2uvrA
0 134 21 13 16 27 20
4 151 18 14 17 31 23
20 160 22 11 8 26 18
100 199 20 11 14 25 19
500 164 17 15 17 27 20
2500 0 (nbl) nbl 0 (ibl) 0 (ibl) 1 (ibl) 1 (ibl)
5000 0 (nbl) 0 (nbl) 0 (nbl) 0 (nbl) 0 (nbl) 0 (nbl)
Positive controls SA SA 9-AA 2-NF 2-NF MNNG
Concentrations (ÎĽg/plate) 1 1 50 5 5 2.5
Mean No. of colonies/plate 601 413 132 192 627 593
S9-Mix With
Test item (µg/plate) TA100 TA1535 TA1537 TA1538 TA98 WP2uvrA
0 143 17 12 13 27 19
4 157 9 11 17 28 16
20 191 12 15 17 35 21
100 193 11 15 17 34 18
500 131 (ibl) 11 16 13 36 19
2500 0 (ibl) 1 (ibl) 0 (ibl) 2 (ibl) 3 (ibl) 7 (ibl)
5000 0 (nbl) 0 (nbl) 0 (nbl) 0 (nbl) 0 (nbl) 0 (nbl)
Positive control 2-AA 2-AA 2-AA 2-AA 2-AA 2-AA
Concentrations (ÎĽg/plate) 0.5 1 1 0.5 0.5 10
Mean No. of colonies/plate 2862 246 463 2520 2740 324
Positive control B(a)P B(a)P B(a)P B(a)P B(a)P B(a)P
Concentrations (ÎĽg/plate) 10 10 10 10 10 10
Mean No. of colonies/plate 635 30 145 245 734 70

ibl: incomplete background lawn in all plates

(ibl): incomplete background lawn in individual plates

nbl: no background lawn

(nbl): no background lawn in individual plates

P: precipitation

SA: sodium azide

9-AA: 9 -aminoacridine

2-NF: 2 -nitrofluorene

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine

2-AA: 2 -aminoanthracen

B(a)P: benzo(a)pyrene

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
26 Feb - 08 Mar 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon and trp operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
Pre-experiment: 4, 20, 100, 500, 2500 and 10000 µg/plate with and without metabolic activation
Main Experiment:
Experiment I:
The pre-experiment served as Experiment I.
Experiment II:
4, 20, 100, 500 and 2500 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
benzo(a)pyrene
other: -S9: 2-nitrofluorene (5 µg/plate) for TA1538 and TA98; N-Methyl-N'-nitro-N-nitrosoguanidine (2.5 µg/plate) for WP2uvrA; +S9: 2-aminoanthracene (0.5, 1 or 10 µg/plate) for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 2 and 3 replications each in 2 independent experiments (Experiment 1 and 2), respectively.

DETERMINATION OF CYTOTOXICITY
- Method: A reduced rate of spontaneously occuring colonies and visible thinning of the bacterial lawn were used as indicator for toxicity.

OTHER EXAMINATIONS:
Sterility check: A sterility check was performed with agar plates only containing the test substance with and without S9 mix.
Statistics:
Mean values were calculated.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1 and 2: ≥ 500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: ≥ 500 µg/plate with and without metabolic activation; Experiment 2: ≥ 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: ≥ 100 µg/plate without metabolic activation and ≥ 500 µg/plate with metabolic activation; Experiment 2: ≥ 500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: ≥ 500 µg/plate with and without metabolic activation; Experiment 2: at 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance on the plates was observed in concentrations ≥ 2500 µg/plate with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment served as Experiment I (all strains were tested in the pre-experiment).

HISTORICAL CONTROL DATA
- Positive historical control data: The historical control data of the positive controls were not provided in the study report, but the control plates with the background control gave the expected number of colonies.
- Negative (solvent/vehicle) historical control data: No historical control data of the vehicle control was provided in the study report, but the control plates with the positive controls gave the expected number of colonies.

OTHER EXAMINATIONS:
Sterility of S-9 Mix and the test compound was indicated by the absence of contamination on the test material and S-9 Mix sterility check plates.

Table 1: Summary of Experiment 1

EXPERIMENT 1
S9-Mix Without
Test item (µg/plate) TA100 TA1535 TA1537 TA1538 TA98 WP2uvrA
0 166 29 14 13 23 24
4 148 31 16 16 22 31
20 160 27 12 13 23 24
100 172 24 15 16 26 25
500 71 (nbl) 17 12 11 (ibl) 29 28
2500 0 P (nbl) P nbl P nbl P nbl 1 (ibl) 2 (ibl)
10000 0 P (nbl) 0 P (nbl) 0 P (nbl) 0 P (nbl) 0 P (nbl) 0 P (nbl)
Positive controls SA SA 9-AA 2-NF 2-NF MNNG
Concentrations (ÎĽg/plate) 1 1 50 5 5 2.5
Mean No. of colonies/plate 469 336 210 2327 574 329
S9-Mix With
Test item (µg/plate) TA100 TA1535 TA1537 TA1538 TA98 WP2uvrA
0 166 15 14 16 27 26
4 169 11 14 18 28 26
20 187 10 16 15 30 24
100 182 15 14 27 47 37
500 149 13 10 21 38 32
2500 P nbl P nbl 0 P (ibl) 0 P (ibl) 3 (ibl) 1 (ibl)
10000 0 P (nbl) 0 P (nbl) 0 P (nbl) 0 P (nbl) 0 P (nbl) 0 P (nbl)
Positive control 2-AA 2-AA 2-AA 2-AA 2-AA 2-AA
Concentrations (ÎĽg/plate) 0.5 1 1 0.5 0.5 10
Mean No. of colonies/plate 658 135 85 535 572 272
Positive control B(a)P B(a)P B(a)P B(a)P B(a)P B(a)P
Concentrations (ÎĽg/plate) 10 10 10 10 10 10
Mean No. of colonies/plate 740 30 143 280 794 60

ibl: incomplete background lawn in all plates

(ibl): incomplete background lawn in individual plates

nbl: no background lawn

(nbl): no background lawn in individual plates

SA: sodium azide

9-AA: 9 -aminoacridine

2-NF: 2 -nitrofluorene

MNNG: N-Methyl-N'-nitro-N-nitrosoguanidine

2-AA: 2-Aminoanthracen

B(a)P: Benzo(a)pyrene

Table 2: Summary of Experiment 2

EXPERIMENT 2
S9-Mix Without
Test item (µg/plate) TA100 TA1535 TA1537 TA1538 TA98 WP2uvrA
0 148 17 10 14 23 23
4 131 15 10 13 23 21
20 141 17 14 14 24 15
100 140 13 12 11 22 22
500 115 14 (ibl) 7 9 21 19
2500 0 P (nbl) P nbl P nbl 0 (nbl) P nbl 1 (ibl)
Positive controls SA SA 9-AA 2-NF 2-NF MNNG
Concentrations (ÎĽg/plate) 1 1 50 5 5 2.5
Mean No. of colonies/plate 505 35 194 935 641 469
S9-Mix With
Test item (µg/plate) TA100 TA1535 TA1537 TA1538 TA98 WP2uvrA
0 157 14 9 21 34 18
4 155 15 9 19 30 22
20 191 12 10 20 37 16
100 176 13 9 18 39 22
500 129 (ibl) 12 (ibl) 10 14 28 18
2500 P nbl P nbl 0 P (nbl) 0 (nbl) 0 (ibl) 0 (ibl)
Positive control 2-AA 2-AA 2-AA 2-AA 2-AA 2-AA
Concentrations (ÎĽg/plate) 0.5 1 1 0.5 0.5 10
Mean No. of colonies/plate 748 147 123 498 546 230
Positive control B(a)P B(a)P B(a)P B(a)P B(a)P B(a)P
Concentrations (ÎĽg/plate) 10 10 10 10 10 10
Mean No. of colonies/plate 829 32 163 259 792 57

ibl: incomplete background lawn in all plates

(ibl): incomplete background lawn in individual plates

nbl: no background lawn

(nbl): no background lawn in individual plates

SA: sodium azide

9-AA: 9 -aminoacridine

2-NF: 2 -nitrofluorene

MNNG: N-Methyl-N'-nitro-N-nitrosoguanidine

2-AA: 2-Aminoanthracen

B(a)P: Benzo(a)pyrene

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Treatment with a mutagen that requires metabolic activation by microsomal enzymes was not used. No strain with an AT base pair at the primary reversion site capable of detection of certain oxidizing mutagens or cross-linking agents was used, e.g. E. coli WP2 or S. typhimurium TA102.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
yes
Remarks:
Treatment with a mutagen that requires metabolic activation by microsomal enzymes was not used. No strain with an AT base pair at the primary reversion site was used, e.g. E. coli WP2 or S. typhimurium TA102.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa-deficient, uvrB-deficient and bio-deficient
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: rfa-deficient, uvrB-deficient and bio-deficient
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
Experiment 1 and 2: 10, 50, 100, 500, 1000 and 2500 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: -S9 and +S9: 2-aminoanthracene (1 or 2.5 µg/plate) for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 1 replication each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: not specified
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Revertants per plate after treatment with the test substance (Experiment 1)

EXPERIMENT 1
S9-Mix Without
Test item (µg/plate) TA100 TA1535 TA1537 TA1538 TA98
10 101 24 5 15 25
50 114 10 5 14 28
100 115 9 5 9 27
500 125 18 5 9 40
1000 110 12 3 5 21
2500 T T T T T
S9-Mix With
Test item (µg/plate) TA100 TA1535 TA1537 TA1538 TA98
10 96 12 12 19 20
50 155 8 7 19 43
100 136 8 9 25 42
500 174 4 9 13 41
1000 129 7 4 8 38
2500 42 7 2 4 9

T: Toxic

Table 2: Revertants per plate after treatment with the test substance (Experiment 2)

EXPERIMENT 2
S9-Mix Without
Test item (µg/plate) TA100 TA1535 TA1537 TA1538 TA98
10 88 31 16 7 36
50 105 26 17 5 41
100 125 19 15 13 31
500 126 19 13 12 38
1000 105 19 6 13 20
2500 T T T T T
S9-Mix With
Test item (µg/plate) TA100 TA1535 TA1537 TA1538 TA98
10 117 6 30 15 39
50 152 12 25 21 45
100 130 8 24 21 64
500 142 8 19 12 27
1000 139 8 17 17 24
2500 56 2 1 11 9

T: Toxic

Table 3: Revertants per plate (Controls of Experiment 1)

  Concentration TA100 TA1535 TA1537 TA1538 TA98
without S9-Mix
Negative Control - 130 17 6 17 33
- 100 29 8 18 26
Sodium azide 1 µg/plate 710 390 - - -
9-Aminoacridine 50 µg/plate - - 425 - -
2-Nitrofluorene 5 µg/plate - - - 686 420
with S9-Mix
Negative Control - 90 12 18 21 49
- 100 14 12 29 32
2-Aminoanthracene 1 µg/plate 1275 - - 468 1395
2.5 µg/plate - 590 200 - -

Table 4: Revertants per plate (Controls of Experiment 4)

  Concentration TA100 TA1535 TA1537 TA1538 TA98
without S9-Mix
Negative Control - 74 19 24 11 32
- 81 18 9 10 36
Sodium azide 1 µg/plate 528 414 - - -
470 359 - - -
9-Aminoacridine 50 µg/plate - - 207 - -
- - 147 - -
2-Nitrofluorene 5 µg/plate - - - 536 313
- - - 587 227
2-Aminoanthracene 1 µg/plate 65 - - 44 33
88 - - 18 49
2.5 µg/plate - 12 13 - -
- 17 26 - -
with S9-Mix
Negative Control - 92 4 24 24 55
- 87 8 32 29 49
2-Aminoanthracene 1 µg/plate 439 - - 246 349
421 - - 213 320
2.5 µg/plate - 171 158 - -
- 165 139 - -
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Dec 23, 1983 - Mar, 07 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
only 100 metaphases per concentration have been evaluated, insufficient substance identity and purity documentation
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. S. Wolff's Laboratory UCSF was the original source. The cells were further cloned at Dr. A. Bloom's Laboratory, Columbia University, New York
- Suitability of cells: This cell line has a high proliferation rate and cloning efficiency. The cells have a stable karyotype and an aberration rate of about 0-5% of the metaphases.
- Methods for maintenance in cell culture if applicable: Large stocks of this clone are stored in liquid nitro gen allowing the repeated use ofthe same cell culture batch in different experiments.
- Modal number of chromosomes: 22 +/- 1
- Normal (negative control) cell cycle time: 12 to 14 hours corresponds to 1.5 normal cell cycle length

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The cells were cultured in supplemented Dulbecco's Minima! Essential Medium (DMEM). The DMEM was supplemented with L-glutamine (4 mM), sodium bicarbonate (0.375 %), antibiotics (neomycine 0.015 %), and 10 % fetal calf serum (FCS). All incubations were performed at +37°C in a 4-5 % carbon dioxide atmosphere (100 % humidity).
- Periodically checked for Mycoplasma contamination: yes
- Other: The cells were cloned to minimize the frequency of mutants, and to stabilize the karyotype. The cells have a spontaneous aberration rate of about 0-5 % aberrant metaphases.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix induced by Aroclor 1254
Test concentrations with justification for top dose:
100, 150, 200, 250, and 300 µg/mL (+/- S9 mix)
The highest concentration of the test item was chosen based on the toxicity of the test item.
Vehicle / solvent:
- Vehicle/solvent used: ethanol
- Justification for choice of solvent/vehicle: solubility performance
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:

DURATION
- Exposure duration: 16.5 hours (- S9 mix); 2 hours (+ S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 16.5 and 20 hours, respectively

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Cells were stained in 5 % Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The medium is replaced by KCl-solution (0.075 %) for hypotonic treatment. For fixation of cells fixative ( methanol: acetic acid, 3: 1) is added and then renewed 2 times.cells were dropped on slides and air dried.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, cell viability

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

The frequnecy determinaton of polyploid cells and endoreduplications (chromosomes with 4, 8, 16 ... chromatids) is based on scoring 100 mitoses per slide, and estimation of the mitotic index on scoring 100 cells per slide.
Evaluation criteria:
Students T test was applied to compare the aberration frequency in treated cells with pooled results from solvnet and negative controls. The difference was was considered significant wher p>0.05.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Precipitation: not reported

Conclusions:
In conclusion, treatment of CHO cell cultures with the test item, did increase the proportion of cells with aberrant chromosomes.The test item was clastogenic in this in vitro test system.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo mammalian chromosome aberration test (according to OECD 475, GLP): negative in bone marrow cells of Chinese hamster

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
other information
Study period:
09 Jul - 08 Aug 1985
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
(only one concentration tested, mortality in 1/5 males and clinical signs of systemic toxicity in 2/10 animals; no clear cytotoxicity observed in the bone marrow based on mitotic index; 1000 polychromatic erythrocytes were counted per animal while 4000 is recommended)
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted Jul 2016
Deviations:
yes
Remarks:
(only one concentration tested, mortality in 1/5 males and clinical signs of systemic toxicity in 2/10 animals; no clear cytotoxicity observed in the bone marrow based on MI; 1000 PCEs were counted per animal while 4000 is recommended)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
NMRI
Remarks:
Hoe: NMRKf (SPF71)
Details on species / strain selection:
The mouse has been chosen for this study since it provides a convenient in vivo mammalian model, which has been proposed in the literature.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: 8 weeks
- Weight at study initiation: Ranges: 28 - 36 g (males); 23 - 29 g (females)
- Housing: Groups of 5 animals were housed in macrolon cages (Type 3) on softwood granulate.
- Diet: Altromin 1324 (Altromin - GmbH, Lage/Lippe, Germay), ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: polyethylene glycol 400
- Concentration of test material in vehicle: 15% (w/v)
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance dilutions were freshly prepared each day. 3750 mg of the test substance was weighed into a beaker. About 15 ml of polyethylenglycol 400 was given into a 25 ml flask. Slowly the substance was given into the flask and solubilized entirely. Then the flask was topped up to the calibration mark with polyethylenglycol 400 and mixed thoroughly until a clear solution was formed.
Duration of treatment / exposure:
not applicable
Frequency of treatment:
single treatment
Post exposure period:
24, 48 or 72 h after last treatment
Dose / conc.:
1 500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals per sex per dose and per post exposure period
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control: Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 50 mg/kg bw in distilled water
Tissues and cell types examined:
Tissue: bone marrow
Cell type: erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on a preliminary study (09 – 21 Jul 1985) for determination of the acute toxicity, the dose levels for micronucleus testing were selected. 3 females were dosed at 1500 and 2500 mg/kg bw, respectively, and 3 males were dosed at 2000 mg/kg bw. All females of the highest dose group and one male of the mid-dose group died. Abdominal position, back-arched position, ptosis and gasping were observed in all dose groups. Tachypnoe was noted only in the low-dose group. Hyperreflexia and piloerection was observed in the low- and mid-dose group. Animals of the mid-dose group showed reduced spontaneous activity. Dopiness was seen in the mid- and high-dose group and increased respiratory sounds occurred in the high-dose group. Therefore the highest possible sublethal dose of 1500 mg/kg bw was selected for the main study.

TREATMENT AND SAMPLING TIMES: Sampling was performed 24, 48 and 72 h after administration.

DETAILS OF SLIDE PREPARATION: Slides were fixed with methanol and stained with May-GrĂĽnwald for 5 min and with Giemsa-solution for 10 min.

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition the ratio of polychromatic to normochromatic erythrocytes was determined.
Statistics:
For evaluation all bone marrow smears are coded to ensure that the group to which they belonged remained unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occuring in 1000 counted polychromatic erythrocytes, and the number of normocytes with micronuclei occuring in 1000 counted normocytes, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).
The results of the test substance at each sampling time and dose were compared with the corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). The statistical evaluations were realized with the "Diamant" computer program Version 2.0, supplied by the Department "Information und Kommunikation" of Hoechst AG. All statistical results are based on the 95 % level of significance.
Key result
Sex:
female
Genotoxicity:
positive
Remarks:
at 1500 mg/kg bw and at all sampling times
Toxicity:
yes
Remarks:
Irregular breathing, disturbance equilibrium, dopiness, hyperreflexia and backed-arched position
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
positive
Remarks:
at 1500 mg/kg bw and at all sampling times
Toxicity:
yes
Remarks:
Irregular breathing, disturbance equilibrium, dopiness, hyperreflexia and backed-arched position; one male died 5 h after application and was replaced
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Results of the in vivo micronucleus assay in male mice

  PCE/NCE Total Number of PCE with MN of 1000 cells PCE with MN/1000 cells
Dose group Number of animals Dose [mg/kg bw] 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h
Vehicle control (polyethylenglycol 400 5 - 1.02 0.94 0.90 1 1 1 0.08 0.08 0.08
Positive control (cyclophosphamide) 5 50 0.65 n.d. n.d. 27 n.d. n.d. 2.74 n.d. n.d.
Test substance 5 1500 0.85 0.83 1.00 3 4 5 0.34 0.38 0.52

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

MN: micronucleus

n.d.: not determined

Table 2: Results of the in vivo micronucleus assay in female mice

  PCE/NCE Total Number of PCE with MN of 1000 cells PCE with MN/1000 cells
Dose group Number of animals Dose [mg/kg bw] 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h
Vehicle control (polyethylenglycol 400 5 0.95 1.00 1.01 1 1 1 0.10 0.10 0.18
Positive control (cyclophosphamide) 5 50 0.71 n.d. n.d. 24 n.d. n.d. 2.44 n.d. n.d.
Test substance 5 1500 0.94 1.09 1.02 2 3 3 0.22 0.34 0.26

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

MN: micronucleus

n.d.: not determined

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Dec 1987 - 06 Jan 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
(2 sampling times instead of the recommended 3; 50 metaphases counted while 200 is recommended; the cell cycle length not given; 500 cells scored for MI while 1000 is recommended; no cytotoxicity observed in bone marrow based on MI)
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Version / remarks:
adopted Jul 2016
Deviations:
yes
Remarks:
(2 sampling times instead of the recommended 3; 50 metaphases counted while 200 is recommended; the cell cycle length not given; 500 cells scored for MI while 1000 is recommended; no cytotoxicity observed in bone marrow based on MI)
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
hamster, Chinese
Strain:
other: Chinese hamster inbred strain
Details on species / strain selection:
The Chinese hamster is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. This species, although little used in other aspects of toxicology, has a karyotype with 22 chromosomes all of which are easily identified. This offers the opportunity to reduce observer errors and the time required for the cytogenetic analysis.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: LMP stock
- Age at study initiation: Minimum 10 weeks
- Weight at study initiation: Approximately 25 g
- Fasting period before study: Yes, animals received no food approximately 18 h beore treatmen with the test substance.
- Housing: The animals were individually housed in Makrolon Type I cages with wire mesh top (EBECO, Castrop-Rauxel, Germany) on granulated soft wood bedding (Altromin, Lage/Lippe, Germany).
- Diet: Pelleted standard diet (Altromin, Lage/Lippe, Germany)
- Water: Tap water, ad libitum
- Acclimation period: Minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Methocel MC
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative nontoxicity for the animals.
- Amount of vehicle: 20 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test substance was suspended in 0.5 % Methocel solution. All animals received a single standard dose volume adjusted to the body weight orally.
Duration of treatment / exposure:
not applicable
Frequency of treatment:
single treatment
Post exposure period:
24 and 48 h after treatment
Dose / conc.:
4 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 30 mg/kg bw in physiological saline
Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A range finding study was performed to find the maximum tolerated dose of the test substance based on mortality. In the first pre-experiment 2 males and 2 females were treated with 5000 mg/kg bw. In the second pre-experiment 10 males were exposed to 5000 mg/kg bw and in the third pre-experiment 2 males and 2 females received 4000 mg/kg bw.

DETAILS OF SLIDE PREPARATION: Following treatment and prior to sample collection, animals were injected intraperitoneally with 2 mg/mL Colcemid®, and samples were collected 2.5 hours thereafter. Cells were harvested from the bone marrow, swollen, fixed and stained, and analysed for chromosomal aberrations.

METHOD OF ANALYSIS: At least 50 well spread metaphases were analysed per animal by use of NIKON microscopes with 100x oil immersion objectives and were scored for structural chromosome aberrations (gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations).
Evaluation criteria:
The test article is classified as mutagenic if it induces a significantly increased aberration rate with at least one of the concentrations tested as compared with the negative control.
This can be confirmed by means of the nonparametric Mann-Whitney test. A test article which produces no significant positive response at any test point is regarded as non-mutagenic in this assay.
Statistics:
Statistical analysis was not performed due to the obtained results.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 4000 and 5000 mg/kg bw
- Solubility: Suspension in 0.5% Methocel solution
- Clinical signs of toxicity in test animals: Reduction of spontaneous activity, apathy and abdominal position were observed in both dose groups. 1/2 males of the first pre-experiment and 5/10 males of the second pre-experiment died at 5000 mg/kg bw. None of the exposed animals died at 4000 mg/kg bw.

Table 1: Summary of results

Dose (mg/kg bw) Postexposure period [h] Number of cells scored Aberrant cells incl. gaps (%) Aberrant cells excl. gaps (%) Mitotic index (%)
4000 24 500 0.6 0.0 5.50
4000 48 500 2.2 0.4 6.51
Negative control 24 500 2.4 1.2 5.60
Positive control 24 500 13.4 9.8 5.28
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Genetic toxicity (mutagenicity) in bacteria in vitro


Ames test


A GLP-conform reliable bacterial gene mutation assay (Ames test) was performed with 2,3,4-trihydroxybenzophenone (CAS 1143-72-2) equivalent or similar to OECD 471 (reference 7.6.1-1). The S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and E. coli WP2uvrA were tested conducting the plate incorporation method in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 2 or 3 replicates each in two independent experiments up to concentrations of 10000 µg/plate in Experiment I and up to concentrations of 5000 µg/plate in Experiment II. Cytotoxicity based on incomplete or no background lawn was observed in all strains from 2500 µg/plate and onwards without metabolic activation. In the presence of S9 mix, cytotoxicity was observed at concentrations ≥ 2500 µg/plate for TA 1535, TA 1537, TA 1538, TA 38 and WP2uvrA, and ≥ 500 µg/plate for TA 100. Precipitation of the test substance on the plates was observed in the highest concentration of 10000 µg/plate with and without metabolic activation. Appropriate positive and solvent controls were included into the test and showed the expected results. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Thus, under the conditions of the study, no mutagenic activity was observed in any strain/activation combination in the bacterial Ames-Test for 2,3,4-trihydroxybenzophenone.


A second GLP-conform Ames test equivalent or similar to OECD 471 was available with 2,3,4-trihydroxybenzophenone (CAS 1143-72-2) (reference 7.6.1-2). The S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and E. coli WP2uvrA were tested conducting the plate incorporation method in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 2 or 3 replicates each in two independent experiments up to concentrations of 10000 µg/plate in Experiment I and up to concentrations of 2500 µg/plate in Experiment II. Cytotoxicity based on incomplete or no background lawn was observed from 2500 µg/plate and onwards in TA 1535, TA 1537, TA 98 and WP2uvrA and from 500 µg/plate and onwards in TA 100 and TA 1538 without metabolic activation. In the presence of S9 mix, cytotoxicity was observed at concentrations ≥ 2500 µg/plate for TA 1535, TA 1537, TA 1538, TA 38 and WP2uvrA, and ≥ 500 µg/plate for TA 100. Precipitation of the test substance on the plates was observed at concentrations ≥ 2500 µg/plate with and without metabolic activation. Appropriate positive and solvent controls were included into the test and showed the expected results. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Thus, under the conditions of the study, no mutagenic activity was observed in any strain/activation combination in the bacterial Ames-Test for 2,3,4-trihydroxybenzophenone.


A third Ames test was conducted with 2,3,4-trihydroxybenzophenone (CAS 1143-72-2) equivalent or similar to OECD 471 (reference 7.6.1-3). The S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were tested by using the plate incorporation method in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 1 replicate each in two independent experiments up to concentrations of 2500 µg/plate in both experiments. Cytotoxicity was observed in all tester strains from 2500 µg/plate and onwards with and without metabolic activation. Appropriate positive and solvent controls were included into the test and showed the expected results, however treatment with a mutagen that required metabolic activation by microsomal enzymes was not used at the study. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Thus, under the conditions of the study, no mutagenic activity was observed in any strain/activation combination in the bacterial Ames-Test for 2,3,4-trihydroxybenzophenone.


 


Cytogenicity was observed in vitro in CHO cells with metabolic activation. No cytogenicity was evident without metabolic activation. However, the study has significant limitations as relevant information is not provided in the report (e.g. substance purity and identity) or the methods employed do not follow the guideline standards (e.g. number of metaphases analysed, number of replicates for negative controls).


 


 


Genetic toxicity (cytogenicity) in vivo mammalian somatic cell study


Micronucleus


Due to methodological deficiencies this study is only regarded as other information with a reliability of 3 (not reliable).


2,3,4-trihydroxybenzophenone (CAS 1143-72-2) was tested in a mammalian erythrocyte micronucleus assay conducted according to OECD guideline 474 and compliant with GLP (reference 7.6.2-1). The maximum tolerated dose (MTD) was determined in a preliminary study, in which 2000 mg/kg bw led to clinical signs and mortality, and 1500 mg/kg bw caused clinical signs. Based on this range finding study, 5 NMRI mice per group were exposed by oral gavage to a dose of 1500 mg/kg bw/day, to the vehicle alone (PEG 400) or to the positive control (50 mg cyclophosphamide per kg bw). The test substance was administered in a single treatment. The frequency of micronucleated polychromatic erythrocytes (% MN PCE) was analysed in the bone marrow, sampled 24, 48 and 72 h after the final administration, respectively. The % MN-PCE values in male and female mice were increased when compared with the values in the vehicle control group at all sampling times. In the main study, clinical signs were observed in all animals at least until 24 h after exposure (males and females), and 1/5 males died (this individual was replaced). The observed clinical signs indicated systemic availability of the test substance. However, no cytotoxicity in the bone marrow was observed based on the mitotic index. Based on the results of the conducted study, 2,3,4-trihydroxybenzophenone (CAS 1143-72-2) induced micronuclei in the polychromatic erythrocytes of the bone marrow of male and female mice treated at 1500 mg/kg bw/day. 


 


Chromosome aberration


An in vivo chromosome aberration test performed in male and female Chinese hamsters with 2,3,4-trihydroxybenzophenone (CAS 1143-72-2) according to OECD TG 475 (dated 1984) and according to GLP was available (reference 7.6.2-2). The use of the hamster (non-standard species) was justified in the study report. A range finding study was performed to find the maximum tolerated dose of the test substance based on mortality. Based on this range finding study, 6 animals per group were exposed by oral gavage to a dose of 4000 mg/kg bw/day, to the vehicle alone (Methocel MC) or to the positive control (30 mg cyclophosphamide per kg bw). In the study, there were 2 sampling times, 24 and 48 h. Furthermore, only 50 metaphases were counted and only 500 cells were scored for determination of the mitotic index. It was unclear if the substance was systemically available and if the bone marrow was exposed, since no cytotoxic effects based on the mitotic index were observed. The included negative and the positive control can be considered as valid. The result of this chromosome aberration study with 2,3,4-trihydroxybenzophenone (CAS 1143-72-2) was negative, since the test substance did not induce the frequency of aberrant cells after both sampling times.

Justification for classification or non-classification

Based on the available information there exist evidence for cytogenicity in vitro. Further results from the in vivo micronucleus test indicate evidence for genotoxicty / clastogenicity in vivo. In addition it should be noted, that the available in vivo data lack the quality standards required for high reliablity, e.g. single dose only and therefore no dose reponse relationsship. As these positive findings are or not supported by data on mutagenicity from in vitro or in vivo assays, the test substance 2,3,4 -trihydroxybenzophenone (CAS 1143 -72 -2), does not meet the classification criteria for Mutagenicity (e.g. Muta 2, H341) according to Regulation (EC) 1272/2008.