Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
MEXORYL SAB didn't induce mutagenicity in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102. It induced chromosome aberrations in CHO cells only in presence of metabolic activation. It was negative in an in vivo micronucleus test with mice and an in vivo UDS test with rats.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study similar to OECD guideline 474 with minor deviations: data about test substance purity, no certificate of analysis, animal bodyweight not followed, standard deviations not reported in the results.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
no data about test substance purity; no certificate of analysis; animal bodyweight not followed; standard deviations not reported in the results
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Limited, Margate, U.K.
- Fasting period before study: no
- Housing: in groups of 5 per cage
- Diet (e.g. ad libitum): SQC rat and mouse maintenance diet no. 1 (Special Diets Services Limited, Witham, England), ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12 h/12 h
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: test substance: carboxymethylcellulose (CMC); positive control: 0.9% sterile saline
- Concentration of test material in vehicle: test substance: 0.5%; positive control: 0.4 mg/mL
Details on exposure:
No data
Duration of treatment / exposure:
Single administration
Frequency of treatment:
Single administration
Post exposure period:
Range finding study: 3 days
Main study: 24, 48 and 72 h
Remarks:
Doses / Concentrations:
250 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C
- Route of administration: intraperitoneal injection
- Doses / concentrations: 4 mg/kg
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on the range finding study where animals exposed at 1000 mg/kg (highest dose tested) had no critical signs of toxicity

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): for each dose group, 5 males and 5 females were sacrified at 24, 48 and 72 h after dosing

DETAILS OF SLIDE PREPARATION:
Marrow cells were centrifuged at 800 rpm for 5 min, the supernatant withdrawn, and the cells re-suspended in a minimal volume of foetal calf serum. One drop of cell suspension was placed on each of 2 slides and was left to air dry for 24 hours before staining based on the method of Gollapudi and Kamra. Cells were fixed for 5 min in methanol, rinsed twice in deionised water, stained for 10 min in Giemsa and rinsed several times in tap water and finally in deionised water.

METHOD OF ANALYSIS: A minimum of 1000 polychromatic erythrocytes (PCE), including micronucleated PCE, were counted for each animal.
Evaluation criteria:
Based on statistical analysis and the toxicity shown by PCE/NCE ratio.
Statistics:
Analysis of micronucleus counts to determine the significance of differences between control and Mexoryl SAB treated groups was carried out by the Mann-Whitney U test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Decrease of PCE/NCE ratio for 1000 mg/kg at 24 h
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 50, 320 and 1000 mg/kg
- Clinical signs of toxicity in test animals: no severe toxic events
- Evidence of cytotoxicity in tissue analyzed: not examined

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): after 24 h at 1000 mg/kg
- Ratio of PCE/NCE (for Micronucleus assay): 0.52 (males and females) at 24 h
- Appropriateness of dose levels and route: acceptable
- Statistical evaluation: Mann-Whitney U test

Table 1: group means for animals sacrificed at 24 h

 

 

Mean

Mean

Mean

Ratio

Mean %

Group

 

PCE

MN-PCE

MN-NCE

PCE/NCE

MN-PCE

CONTROL

M

1031.4

0.0

0.0

0.83

0.00

CONTROL

F

1004.2

0.0

0.0

0.88

0.00

CONTROL

M+F

1017.8

0.0

0.0

0.86

0.00

 

 

 

 

 

 

 

250 mg/kg

M

1018.4

0.0

0.0

1.06

0.00

250 mg/kg

F

1047.2

0.2

0.0

1.26

0.02

250 mg/kg

M+F

1032.8

0.1

0.0

1.16

0.01

 

 

 

 

 

 

 

500 mg/kg

M

1027.8

0.0

0.0

1.08

0.00

500 mg/kg

F

1023.2

0.0

0.0

1.14

0.00

500 mg/kg

M+F

1025.5

0.0

0.0

1.11

0.00

 

 

 

 

 

 

 

1000 mg/kg

M

1018.2

0.0

0.2

0.60

0.00

1000 mg/kg

F

1036.0

0.2

0.0

0.44

0.02

1000 mg/kg

M+F

1027.1

0.1

0.1

0.52

0.01

 

 

 

 

 

 

 

Mitomycin C

M

1024.2

8.4

0.2

1.23

0.82

Mitomycin C

F

1064.8

12.6

0.6

1.22

1.19

Mitomycin C

M+F

1044.5

10.5

0.4

1.23

1.01

Table 2: group means for animals sacrificed at 48 h

 

 

Mean

Mean

Mean

Ratio

Mean %

Group

 

PCE

MN-PCE

MN-NCE

PCE/NCE

MN-PCE

CONTROL

M

1006.2

0.0

0.0

1.26

0.00

CONTROL

F

1018.4

0.0

0.0

1.44

0.00

CONTROL

M+F

1012.3

0.0

0.0

1.35

0.00

 

 

 

 

 

 

 

250 mg/kg

M

1010.8

0.0

0.0

0.97

0.00

250 mg/kg

F

1034.0

0.0

0.0

0.90

0.00

250 mg/kg

M+F

1022.4

0.0

0.0

0.93

0.00

 

 

 

 

 

 

 

500 mg/kg

M

1011.4

0.0

0.0

1.05

0.00

500 mg/kg

F

1053.0

0.0

0.0

1.06

0.00

500 mg/kg

M+F

1032.2

0.0

0.0

1.06

0.00

 

 

 

 

 

 

 

1000 mg/kg

M

1041.8

0.0

0.0

1.14

0.00

1000 mg/kg

F

1046.6

0.0

0.0

1.27

0.00

1000 mg/kg

M+F

1044.4

0.0

0.0

1.21

0.00

Table 3: group means for animals sacrificed at 72 h

 

 

Mean

Mean

Mean

Ratio

Mean %

Group

 

PCE

MN-PCE

MN-NCE

PCE/NCE

MN-PCE

CONTROL

M

1006.2

0.0

0.0

1.26

0.00

CONTROL

F

1018.4

0.0

0.0

1.44

0.00

CONTROL

M+F

1012.3

0.0

0.0

1.35

0.00

 

 

 

 

 

 

 

250 mg/kg

M

1010.8

0.0

0.0

0.97

0.00

250 mg/kg

F

1034.0

0.0

0.0

0.90

0.00

250 mg/kg

M+F

1022.4

0.0

0.0

0.93

0.00

 

 

 

 

 

 

 

500 mg/kg

M

1011.4

0.0

0.0

1.05

0.00

500 mg/kg

F

1053.0

0.0

0.0

1.06

0.00

500 mg/kg

M+F

1032.2

0.0

0.0

1.06

0.00

 

 

 

 

 

 

 

1000 mg/kg

M

1041.8

0.0

0.0

1.14

0.00

1000 mg/kg

F

1046.6

0.0

0.0

1.27

0.00

1000 mg/kg

M+F

1044.4

0.0

0.0

1.21

0.00

Conclusions:
Interpretation of results (migrated information): negative
Under these test conditions, Mexoryl SAB was found not to be clastogenic in vivo on bone marrow cells of mice.
Executive summary:

In an in vivo micronucleus test similar to OECD guideline 474 and in compliance with GLP, mice were exposed to Mexoryl SAB. In a range finding assay, pairs of male mice were orally exposed to Mexoryl SAB at concentrations of 50, 320 and 1000 mg/kg without any critical sign of toxicity. Then, 5 animals/sex/dose/time of sacrifice were given single dose of 250, 500 and 1000 mg/kg of Mexoryl SAB by oral gavage and were sacrificed at 24, 48 and 72 h after dosing. Then bone marrow cells were harvested, stained with Giemsa and analysed for micronuclei.

Positive controls (mitomycin C at 4 mg/kg intraperitoneally) induced an appropriate increase in the number of polychromatic erythrocytes. Micronuclei were not induced at any tested concentrations and at any time of sacrifice in control and in Meroxyl SAB-treated mice despite a decrease in polychromatic erythrocytes/normochromatic erythrocytes for 1000 mg/kg at 24 h.

Under the test conditions, Mexoryl SAB was found not to be clastogenic in vivo in mice.

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

Additional information

Additional information from genetic toxicity in vivo:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD guideline 471, in compliance with GLP, MEXORYL SAB was not mutagenic in S. typhimurium (TA 1535, TA 1537, TA 1538, TA 100 and TA 98) and one Escherichia coli strain (WP2 uvrA).

In an in vitro chromosome aberration test performed in Chinese Hamster Ovary (CHO) cells according to OECD guideline 473 and in compliance with GLP, MEXORYL SAB induced an increase in chromosome aberrations only in presence of metabolic activation.

In an in vivo micronucleus test performed in mice, similar to OECD guideline 474 and in compliance with GLP, micronuclei were not induced at any tested concentrations and at any time of sacrifice in control and in MEXORYL SAB-treated mice despite a decrease in polychromatic erythrocytes/normochromatic erythrocytes for 1000 mg/kg at 24 h.

Also, in a GLP study conducted in compliance with OECD guideline 486, MEXORYL SAB did not induce unscheduled DNA synthesis in primary rat hepatocytes after in vivo treatment up to 2000 mg/kg bw.

Justification for classification or non-classification

Despite an induction of chromosome aberrations in presence of metabolic activation, MEXORYL SAB was negative in two in vivo tests (micronulceus test in mice and UDS in rats hepatocytesafter in vivo treatment) and was not mutagenic in several bacterial strains. Therefore, MEXORYL SAB is not classified as mutagenic according to Directive 67/548/EEC and in CLP Regulation.