7-hydroxycitronellal

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 2017 - May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

Jul 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

7-hydroxycitronellal

EC Number:

203-518-7

EC Name:

7-hydroxycitronellal

Cas Number:

107-75-5

Molecular formula:

C10H20O2

IUPAC Name:

7-hydroxy-3,7-dimethyloctanal

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material: 00084577L0
- Purity: 99.4%
- Physical state, appearance: liquid, colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: guaranteed
- Homogeneity: guaranteed, ensured by mixing

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: weighed, topped up and dissolved in culture medium to achieve the required concentration. Achieving a solution, ultrasonic waves, shaking or pipetting thoroughly was used. Solutions were prepared immediately before administration.

OTHER SPECIFICS:
- measurement of pH (at least top concentrations and negative control), osmolality (at least top concentration and negative control), solubility, cell morphology, and precipitation

Method

Target gene:

X-linked hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: rat liver induced phenobarbital and beta-naphthoflavone
- method of preparation of S9 mix: according to Ames et al. ( 1975)
- concentration or volume of S9 mix and S9 in the final culture medium: 1 part S9 fraction with 9 parts S9 supplement. S9 mix consisted of 10% S9 fraction.

Test concentrations with justification for top dose:

1st experiment: 400, 600, 800, 1000, 1200, 1400, 1600 µg/ml (without S9); 37.5, 75, 150, 300, 600, 800, 1200 µg/ml (with S9)
2nd experiment: 800, 1000, 1200, 1300, 1400, 1500, 1600, 1750 µg/ml (without S9); 200, 400, 800, 1000, 1200, 1300, 1500 µg/ml (with S9)
3rd experiment: 1000, 1200, 1300, 1400, 1500, 1600, 1750 µg/ml (without S9); 200, 400, 800, 1000, 1200, 1300, 1500 µg/ml (with S9)
4th experiment: 200, 400, 800, 1000, 1200, 1300, 1400, 1500, 1600 µg/ml (without S9)
5th experiment: 800, 1000, 1100, 1200, 1250, 1300, 1350, 1400, 1450, 1500 µg/ml (without S9)
The doses/concentrations tested in this study were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test.

Vehicle / solvent:

- Vehicle used: culture medium (Ham's F12)
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, culture medium (Ham's F12) was used as most suitable vehicle.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single
- Number of independent experiments : 5

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 20x10^6 cells in 40 ml
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Test substance incubation: 20-24 h after seeding
- Exposure duration/duration of treatment: 4 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7-9 days
- Selection time (if incubation with a selective agent): 6-7 days, seeded in 20 ml selection medium (Ham's F12 with 6-thioguanine (10 µg/ml) and 10% FCS)
- Fixation time: Fixed with methanol, stained with Giemsa

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS), cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: mutant frequency

- OTHER:
In the 2nd Experiment strong precipitation occurred from 1200.0 μg/mL onward with and without S9 mix while no precipitation could be observed in the pre-test and in the 1st Experiment. Due to this discrepancy, the 2nd Experiment was discontinued and is not reported. This experimental part was repeated in Experiment 3.
In the 3rd Experiment after 4 hours treatment in the absence of metabolic activation strong cytotoxicity occurred at 1300.0 μg/mL and above. Thus, with only two remaining test groups this experimental part of the study did not fulfill the recommendations of the current OECD Guideline 476 and is not reported. This experimental part was repeated in Experiment 4. The results of the experiments 1 and 4 in the absence of metabolic activation were reassessed in an additional experiment (5th Experiment).

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative control value and the range of our laboratory’s historical negative control data (95% control limit)
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit)

Statistics:

Trend test (MS Excel function RGP) assessing possible dose-related increase of mutant frequencies. Statistical significance was judged if trend was below probability value (p value) of 0.05 and slope was greater than 0.
Pair-wise comparison was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

At 1300 µg/ml a statistically increase in mutant frequency was observed but was considered as biologically irrelevant, because the value was well within the historical negative control data range

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1200 µg/ml and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1100 µg/ml and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Not influenced by test substance treatment
- Data on osmolality: Not influenced by test substance treatment
- Precipitation: no precipitation was observed
- Cell morphology: cell morphology and attachment of cells not adversely influenced, except in experiments 4 and 5 after 4 h in the absence of metabolic activation in at least the highest applied concentration where cells were adversely influenced

RESULTS OF MAIN STUDY:
- Summary table of results obtained in the study were attached to dossier.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: range 42.47 - 419.90 (without S9, EMS), mean 158.01, SD 80.87, 95% control limits 0.00 (lower) and 320.94 (upper); range 21.52 - 270.48 (with S9, DMBA), mean 125.89, SD 56.16, 95% control limits 13.02 (lower) and 238.77 (upper)
- Negative historical control data: range 0.00 - 7.09 (without S9), mean 2.86, SD 1.81, 95% control limits 0.00 (lower) and 6.49 (upper); range 0.00 - 9.93 (with S9), mean 2.93, SD 2.24, 95% control limits 0.00 (lower) and 7.43 (upper)

Applicant's summary and conclusion

Conclusions:

Thus, the conclusion is drawn that in the absence of metabolic activation, the test substance induces gene mutations in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Five independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following concentrations were tested, whereby the highest tested concentration. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix

0; 400.0; 600.0; 800.0; 1000.0; 1200.0; 1400.0; 1600.0 μg/mL

with S9 mix

0; 37.5; 75.0; 150.0; 300.0; 600.0; 800.0; 1200.0 μg/mL

2nd Experiment (not valid; data not shown)

without S9 mix

0; 800.0; 1000.0; 1200.0; 1300.0; 1400.0; 1500.0; 1600.0; 1750.0 μg/mL

with S9 mix

0; 200.0; 400.0; 800.0; 1000.0; 1200.0; 1300.0; 1500.0 μg/mL

3rd Experiment

without S9 mix (did not fulfil the recommendations of the OECD Guideline 476; data

not shown)

0; 1000.0; 1200.0; 1300.0; 1400.0; 1500.0; 1600.0; 1750.0 μg/mL

with S9 mix

0; 200.0; 400.0; 800.0; 1000.0; 1200.0; 1300.0; 1500.0 μg/mL

4th Experiment

without S9 mix

0; 200.0; 400.0; 800.0; 1000.0; 1200.0; 1300.0; 1400.0; 1500.0; 1600.0 μg/mL

5th Experiment

without S9 mix

0; 800.0; 1000.0; 1100.0; 1200.0; 1250.0; 1300.0; 1350.0; 1400.0; 1450.0; 1500.0 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week.

Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.

Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-

anthracene (DMBA), led to the expected statistically significant increase in the frequencies of

forward mutations.

In this study, in all experimental parts, at least the highest concentrations evaluated for gene mutations were clearly cytotoxic in the absence and the presence of metabolic activation.

Based on the results of the present study, the test substance caused a statistically significant increase in the mutant frequencies in the absence of metabolic activation in all experiments.

Thus, under the experimental conditions of this study, the test substance is considered to induce forward mutations in the HPRT locus assay under in vitro conditions in CHO cells in the absence of metabolic activation.