Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The study records on genetic toxicity of butyl methoxydibenzoylmethane (BMDBM) comprise two in vitro tests and one in vivo micronucleus test.

In vitro gene mutation study in bacteria
Dr. Dr. Ming Tzan-King 2000 (key): BMDBM showed no mutagenic effect in the Ames test (7 strains, with/without S9 metabolic activation).

In vitro gene mutation study in mammalian cells
Strobel 1984: BMDBM showed no mutagenic effect in the HGPRT test (with/without S9 metabolic activation)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Comparable to guideline study.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM medium + 10 % FCS
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction obtained from Aroclor 1254-induced male Sprague-Dawley rats.
Test concentrations with justification for top dose:
5, 10, 15, 20 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
20 µg/mL reduced the plating efficiency to 12.8 %
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Table 1: Mutagenicity and toxicity data (experiment 1)

 

Concentration [µg/mL]

S9 mix

Mean number of mutant colonies

Mutant colonies per 106cells

Plating efficiency [%]

Negative control

0.0

-

+

3.6 ± 0.9

4.6 ± 1.8

12.8

17.7

67.1

65.7

Negative control + solvent

0.0

-

+

3.4 ± 1.8

3.8 ± 1.3

9.7

18.8

70.2

65.6

Positive control (EMS)

1000

-

73.6 ± 12.3

220.3

71.

Positive control (DMBA)

15.4

+

51.6 ± 2.5

318.9

60.9

Test compound

5.0

-

+

4.8 ± 2.7

4.8 ± 1.8

17.7

19.5

68.7

65.1

 

10.0

-

+

3.8 ± 2.2

6.7 ± 2.3

15.4

27.7

62.4

69.6

 

15.0

-

+

8.6 ± 1.1

3.3 ± 2.1

38.3

12.8

52.3

67.0

 

20.0

-

+

4.0 ± 1.7

5.4 ± 1.5

15.3

21.1

37.6

68.6

 

Table 2: Mutagenicity and toxicity data (experiment 2)

 

Concentration [µg/mL]

S9 mix

Mean number of mutant colonies

Mutant colonies per 106cells

Plating efficiency [%]

Negative control

0.0

-

+

1.6 ± 0.5

2.6 ± 1.7

4.3

11.3

66.7

64.4

Negative control + solvent

0.0

-

+

4.8 ± 1.1

3.8 ± 1.6

12.9

9.9

68.2

69.3

Positive control (EMS)

1000

-

64.4 ± 12.2

190.3

67.2

Positive control (DMBA)

15.4

+

28.4 ± 5.2

105.0

53.5

Test compound

5.0

-

+

7.4 ± 2.9

6.0 ± 2.9

17.8

22.5

64.1

62.7

 

10.0

-

+

4.8 ± 1.5

1.8 ± 0.8

10.8

8.4

51.8

60.3

 

15.0

-

+

2.4 ± 1.3

3.6 ± 0.9

6.9

11.7

50.0

63.8

 

20.0

-

+

4.0 ± 2.9

0.6 ± 0.9

10.8

2.1

44.6

52.9

 

 

Conclusions:
It is concluded that under the experimental conditions described in the report neither butyl methoxydibenzoylmethane (BMDBM) per se nor one of its metabolites formed by rat liver enzymes induced mutations at the HGPRT locus in Chinese hamster cells in vitro.
Executive summary:

The test compound butyl methoxydibenzoylmethane (BMDBM) was assessed for mutagenic properties in two independent experiments in the HGPRT-test with V79 cells. In a pre-experiment concentrations of the test compound solubilised in methanol higher than 20 µg/mL lead to precipitation in the nutrient medium. However, 20 µg/mL reduced the plating efficiency (PE; = survival) to 12.8 % in the absence of S9 mix. In the presence of S9 mix the test compound was only slightly toxic: PE = 91.2 %. With the four concentrations up to 20 µg/mL no indication of mutagenic activity was recorded, neither in the presence nor in the absence of S9 mix. The variations of the mutant clonies/106 cells in both experiments do not show any tendency with regard to an enhancement of mutation rates.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March, 28th - June, 23rd 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to the OECD Guideline 471.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon (Ames et al., 1975)
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:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 supernatant fraction from Sprague-Dawley male rats (8 - 10 weeks old) induced with Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
50, 150, 500, 1500, 5000 µg/plate with metabolic activation
5, 15, 50, 150, 500, 1500, 5000 µg/plate without metabolic activation
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
only DMSO as test solution
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene in H2O
Details on test system and experimental conditions:
1. BACTERIA
Frozen permanents of the tester strains are stored in liquid nitrogen. They are prepared from fresh overnight cultures to which 8 % DMSO is added as a cryoprotective agent.
From the permanents master plates are prepared on Vogel-Bonner (VB) minimal-medium plates enriched with histidine and biotin. Ampicillin is added to the plates used for strains with the R-factor. Master plates are used as a source of bacteria for inoculating the overnight cultures. Master plates are always kept at 4°C and discarded after one month or sooner if the number of spontaneous revertants per plate falls out of the normal range specific for a strain.
Tester strain cultures are grown in Oxoid nutrient broth No. 2 or Difco bacto nutrient broth on a shaker (12 - 13 h, 37 °C) to a density of 1-3 x to 9 cells per mL. Only fresh cultures are used for mutagenicity assays.

2. METABOLIC ACTIVATION SYSTEM
The rat liver homogenate fractions were prepared in the testing laboratory in Kirchzarten, Germany. The preparation of the 9000 g supernatant of liver homogenates (S9) from Sprague-Dawley male rats (8 - 10 weeks old) induced with Aroclor 1254 (500 mg/kg body weight) is in accordance with the method recommended by Ames et al (1975).
Each S9 lot was tested for :
* protein concentration (Lowry Method)
* ability of enzymatic stimulation of benzo(a)pyrene and 2-aminoanthracene induced mutagenesis with tester strains TA98 and TA100.
* sterility
The S9 preparations (in 0.15 M KCl) are stored in liquid nitrogen.
S9-mix containing 10 % S9 is freshly prepared for each mutagenicity assay. The concentration of the cofactors in the S9-mix (per mL) are: distilled water, 0.335 mL; phosphate buffer (0.2 M, pH 7.4), 0.5 mL; NADP (0.1 M), 0.04 mL; glucose-6-phosphate (1 M), 0.005 mL; MgCI2/KCl (0.4 M/1.65 M), 0.02 mL; S9 fraction, 0.1 mL.

3. TOP AGAR
Autoclaved top agar containing 0.6 % Difco agar and 0.5 % NaCl is melted and 10 % of a sterile solution of 0.5 mM L-histidine-HCI / 0.5 mM biotin are added and thoroughly mixed prior to the main test procedure. The molten agar is kept at 45 °C in portions of 2 mL in small sterile tubes.

4. TEST PROCEDURE
The tests is performed using the plate incorporation method.
To each tube containing 2 mL molten top agar 0.1 mL of bacteria is added followed by the test solution (dissolved in H2O or DMSO) and 0.5 mL of S9-mix or phosphate buffer in the assays without metabolic activation. The test components are mixed with a vortex and immediatly poured onto coded minimal agar plates and carefully spread to achieve a uniform distribution of the top agar on the surface of the plate. The mixing, pouring, and distribution is done as quickly as possible. The minimal agar plates contain 20 mL of 1.5 % Difco-Bacto agar in VB medium E with 2 % glucose. Plates are kept for 72 h at 37 °C in the dark and then examined. Besides the counting of the number of revertant colonies (his revertants), the plates are examined for the existence of a normal background lawn and/or precipitates and microscopically for microcolony growth.

5. DOSE LEVELS
An initial test is performed to find a suitable dose range. This will be done with the strain TA100 with and without S9-mix. The following 7 dosages are tested in half-log dilutions, starting with the maximum dose of 5.000 µg/plate using the method of the plate incorporation test as described before. After the incubation of the plates for 48 or 72 h (37 °C) the number of revertant colonies will be noted and the quality of the background lawn of auxotrophs careflilly examinei. The maximum concentration not inhibiting the growth of prototrophs and auxotrophs will be the maximum dose in the main test. If toxicity is not observed 5.000 µg per plate will be the maximum dose. For each exposure level, tester strain and activation system plating will be performed in triplicate.

6. CONTROLS
The solvent used for the test substance will be used as the negative control and plated with each strain in triplicate with and without S9-mix. Control without solvent will also be done in the same manner.
The following chemical mutagens will serve as positive control substances.

7. CONFIRMING GENOTYPES OF TESTER STRAINS
Tests for the genotypes of the strains are included in each mutagenicity assay and comprise:

8. REPETITION
The experiment will be repeated in full after an interval of at least 3 days.
Evaluation criteria:
EVALUATION
There are several criteria for determining a positive result, such as a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system. Biological relevance of the result will be considered first. Statistical methods will be used as an aid in evaluating the test results. However, statistical significance will not be the only determining factor for a positive response
INTERPRETATION OF RESULTS
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested species.
Statistics:
Data are evaluated by an appropriate statistical method (X2, Mohn and Ellenberger, 1977)
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed at 1500 and 5000µg/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:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed at 1500 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed at 1500 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed at 1500 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed at 1500 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
The test item is not mutagenic under the described experimental conditions to Salmonella typhimurium strains TA1535, TA 1537, TA 1538, TA 98, TA100 and TA102 in the presence and absence of a metabolizing system.
Executive summary:

The mutagenicity of the substance was studied with six mutant strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98, TA100, and TA102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pre-treated male rats as metabolic activation system. The test item was dissolved in DMSO and tested in concentrations of 15 to 5000 µg per plate in the presence and of 5 to 5000 µg per plate in the absence of S9. In the presence of S9-mix test item was bacteriotoxic towards the strain TA102 at 1500 µg/plate and in the absence at 500 µg/plate. With the other strains bacteriotoxicity was not observed. Precipitation of the test compound on the plates was observed at 1500 and 5000 µg/plate. Sodium azide, 2-nitrofluorene, 9-aminoacridine, mytomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system. In the concentration range investigated, the test item did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that the test item under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo cytogenicity study
Hugentobler 1989 (key): BMDBM induced neither chromosome breaks nor mitotic non-disjunctions in mouse bone marrow cells after twofold oral application of up to 5000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Comparable to guideline study under GLP
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
: other positive control as recommended, only 3 animals per dose group
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
other: Füllinsdorf Albino SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Institute of Biological and Medical Research, CH-4414 Füllinsdorf, Switzerland
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: 27.8 - 32.1g
- Housing: maximum 6 animals per cage
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: unspecified
Vehicle:
rape oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the test compound was dissolved in rape oil and then administered at 0.1 mL/kg bw.
Duration of treatment / exposure:
The substances were administered 30, respectively 6 h before animals were killed.
Frequency of treatment:
The substances were administered 30, respectively 6 h before animals were killed.
Post exposure period:
No post-observation was performed.
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Basis: nominal in diet
Dose / conc.:
2 500 mg/kg bw/day (nominal)
Remarks:
Basis: nominal in diet
Dose / conc.:
5 000 mg/kg bw/day (nominal)
Remarks:
Basis: nominal in diet
No. of animals per sex per dose:
3 male and 3 female animals were treated per dose group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Procarbazine hydrochloride in PBS
- Route of administration: oral
- Doses / concentrations: 50 mg/kg bw at 0.1 mL/kg bw
Tissues and cell types examined:
bone marrow derived polychromaticerythrocytes
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
Four bone-marrow smears per animal were prepared according to the method of Schmid (Mutat. Res. 1975, 31:9)

METHOD OF ANALYSIS:
For each animal 2000 polychromatic erythrocytes were examined in a double blind fashion. Only cells with clearly identifiable anomalies were recorded as cells containing micronuclei. Micronuclei contained in mature erythrocytes were recorded separately, but not taken into the statistical evaluation.
Evaluation criteria:
For each animal 2000 polychromatic erythrocytes were examined in a double blind fashion. Only cells with clearly identifiable anomalies were recorded as cells containing micronuclei. Micronuclei contained in mature erythrocytes were recorded separately, but not taken into the statistical evaluation.
Statistics:
The results were evaluated by means of the Jonckheere-test and the U-test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Table 1: Micronucleus Test with Fuellinsdorf Albino Mice (SPF) given twofold oral application of BMDBM

Single dose [mg/kg bw]

Animal No.

A: Number of PCE with one or more micronuclei

Median of A

B: Percentage of PCE with one or more micronuclei

Median of B + significance levels

0

1

2

3

5

6

7

6

5

4

12

3

4

4.5

0.30

0.30

0.20

0.60

0.15

0.20

0.23

1000

1

2

3

5

6

7

8

9

7

1

4

3

5.5

0.40

0.45

0.35

0.05

0.20

0.15

0.28

2500

1

3

4

5

6

7

13

4

2

3

10

6

5.0

0.65

0.20

0.10

0.15

0.50

0.30

0.25

5000

3

5

6

7

9

11

3

5

74

4

6

4.5

0.65

0.20

0.10

0.15

0.50

0.30

0.23

Number of PCE scored per animal: 2000

Footnote for significance levels

* Jonckheere <5 %

** Jonckheere <1 %

 

Table 2: Micronucleus Test with Fuellinsdorf Albino Mice (SPF) given twofold oral application of positive control substance Procarbazine HCl

Single dose [mg/kg bw]

Animal No.

A: Number of PCE with one or more micronuclei

Median of A

B: Percentage of PCE with one or more micronuclei

Median of B + significance levels

0

1

2

3

5

6

7

6

5

4

12

3

4

4.5

0.30

0.30

0.20

0.60

0.15

0.20

0.23

50

1

2

3

98

68

89

89

4.90

3.40

4.45

4.45

Number of PCE scored per animal: 2000

Footnote for significance levels

* Jonckheere <5 %

** Jonckheere <1 %

Conclusions:
After twofold oral application of 1000, 2500 and 5000 mg/kg bw, butyl methoxydibenzoylmethane induces neither chromosome breaks nor mitotic non-disjunctions in bone marrow cells of mice.
Executive summary:

Butyl methoxydibenzoylmethane (BMDBM), an UVA sunscreen, was evaluated for a potential induction of chromosome breaks and/or mitotic non-disjunctions in vivo by means of the micronucleus test.

After twofold oral application of 1000, 2500 and 5000 mg BMDBM per kg body-weight 30 and 6 hours prior to sacrifice of the mice, no compound related increase of micronuclei could be observed. The dose of 5000 mg/kg is the highest applicable one as determined by preliminary experiments.

It is therefore concluded that under the experimental conditions described in this report, BMDBM induces neither chromosome breaks nor mitotic non-disjunctions in mouse bone marrow cells.

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

Additional information

As in different assays on cytogenicity and mutation, no indications for any genetic toxicity was observed, it can be concluded that BMDBM is not genotoxic in vitro or in vivo.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008T

he available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.