Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-12-02 to 2021-04-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)

EC Number:

931-276-9

Cas Number:

114959-46-5

Molecular formula:

See information in Section 1.2.

IUPAC Name:

Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): [CAS Number 114959-46-5]
- Physical state: Yellow-brown viscous liquid
- Lot/batch No.: E01324-237
- Expiration date of the lot/batch: 2022-10-13
- Storage condition of test material: Room temperature

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 13 October 2022
- Purity: 29.7 %
- Correction factor: 3.37

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 167 ± 6.7 g (mean); 133 – 200 g (range)
- Assigned to test groups randomly: Yes
- Housing: Polycarbonate cages (Makrolon type IV, height 18 cm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Colour-coded cage card indicating Test Facility Study No., group, animal number(s).
- Diet (e.g. ad libitum): Pellets; ad libitum, except during designated procedures. SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
- Water (e.g. ad libitum): Municipal tap water; ad libitum via water bottles
- Acclimation period: At least 5 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 20.1 °C
- Humidity (%): 43 to 51 %
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 07 December 2020 To: 07 January 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A correction factor of 3.37 for the purity/composition of the test item was applied in this study. The test item was dissolved in corn oil. The density of vehicle is 0.9 g/mL. Test item concentrations were dosed within 3 hours after preparation. The dosing volume was 10 mL/kg body weight.

In the dose-range finding the test item was stirred for 30 minutes at 60°C to assure homogeneity.

Frequency of treatment:

Twice with a 24-hour interval

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 19 mg/kg bw/day

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes from bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE/SEX SELECTION:
Selection of an adequate dose-range for the micronucleus main test was based on a dose-range finding study. The test procedure and conditions were similar to those applied in the main test.

One dose group, comprising of 3 males and 3 females were dosed with the test item at the highest concentration that was used for the main study. The observation period after dosing was two days. During this period mortality and physical condition were recorded at least once a day.

Based on the results of the dose-range finding test a full study with one sex was performed. Since there were no substantial differences in toxicity between sexes, only male animals were used in the main study.

TREATMENT AND SAMPLING TIMES:
Bone marrow was sampled 48 hours after the first dosing. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 4 mL of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 216 g for 5 min.

DETAILS OF SLIDE PREPARATION:
The supernatant was removed with a Pasteur pipette. Approximately 500 µL serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100 % methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.

The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, The Netherlands). This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated cover slipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

METHOD OF ANALYSIS:
To prevent bias, all slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5 %). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated. Parts on the slides that contained mast cells that might interfere with the scoring of micronucleated polychromatic erythrocytes were not used for scoring.

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95 % control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data will be analysed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).

A test item is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95 % control limits of the historical control data range.

A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95 % control limits of the negative historical control data range.

Statistics:

ToxRat Professional v3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The animals of the groups treated with 500 mg test item/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. Clinical observations were made in the groups treated with 1000 and 2000 mg test item/kg body weight, these included diarrhea (mid and high dose) and swollen abdomen (high dose).

The mean number of micronucleated polychromatic erythrocytes scored in test item treated groups were compared with the corresponding solvent control group. No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test item compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95 % control limits of the distribution of the historical negative control database.

Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95 % control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

The animals of the groups, which were treated with the test item and the vehicle control showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test item on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Applicant's summary and conclusion

Conclusions:

In conclusion, the test item is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described.

Executive summary:

The objective of the study was to obtain information on the clastogenicity and aneugenicity of the test item when administered to rats at a maximum required acute dose, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow. The study procedures described are in compliance with the most recent OECD and EC guidelines.

 

The test item was a yellow-brown liquid, which was dissolved in corn oil. Based on the results of the dose-range finding study, test concentrations of 2000 mg/kg/day for male animals was selected as the maximum dose for the main test (the highest dose required in the current guideline). Since there were no substantial differences in toxicity between sexes only males were used in the main study.

 

In the main study male animals were dosed twice by oral gavage with vehicle or with 2000, 1000 and 500 mg test item per kg body weight. A positive control group was dosed once by oral gavage with 19 mg cyclophosphamide (CP) per kg body weight. In total 5 treatment groups were used, each consisting of 5 animals. The animals showed the following toxic signs after dosing: diarrhea (mid and high dose) and swollen abdomen (high dose). No mortality was noted in any animal treated with the test item or control animals receiving vehicle or cyclophosphamide.

 

Bone marrow was sampled 48 hours after the first dosing. No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test item compared to the vehicle treated animals.

 

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of negative control animals was within the 95 % control limits of the distribution of the historical negative control database. Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95 % control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

 

The groups that were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

 

In conclusion, the test item is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.