2-hydroxy-5-octanoylbenzoic acid

Endpoint summary

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

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 or test system 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.