7-hydroxycitronellal

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation:

- Ames equivalent to OECD 471, GLP (unspecified), TA 98, TA 100, TA 1535, TA 1537, TA 1538, with and without S9, up to 3.6 mg/plate, negative genotoxicity (Wild et al., 1983)

- HPRT according to OECD 476, GLP, CHO cells, 200 -1600 µg/ml, with and without S9, positive genotoxicity (BASF SE, 2018)

Link to relevant study records

Reference

Reference 1

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

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

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

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

Target gene:

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

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.

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.

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

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)

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable publication which meets basic scientific principles, limited documentation (results in summary table only)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

e.g. strains used

Principles of method if other than guideline:

acc. to Ames B. N ., McCann J . & Yamasaki E . (1975) . Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test . Mutation Res . 31, 347 .

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 mix

Test concentrations with justification for top dose:

up to 3.6 mg/plate

Vehicle / solvent:

No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: 0.5 µg/plate; for TA 1535, TA 100

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 5 µg/plate; T1 100, TA 1537, TA 1538, TA 98

Details on test system and experimental conditions:

METHOD OF APPLICATION: standard plate test, preincubation test (not specified for hydroxycitronellal)


DURATION
- Preincubation period: 20 min, 37°C
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: at least 2

Evaluation criteria:

Positive, if a reproducible, dose-related and at least two-fold elevation of the spontaneous revertant frequency was observed
Agents producing reproducible, dose-related and significant but less than two-fold elevations were classified as marginally mutagenic under the experimental conditions.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Resutls given in tabular form only.

A structurally and metabolically related substance was tested under comparable testing conditions:

Hydroxycitronellol (CAS. 107-74-4): negative

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Equivalent to OECD 474, GLP (unspecified), NMRI mice, intraperitoneal, 345, 603, 861 mg/kg bw, not cytogenic in mice (Wild et al., 1983)

Testing Poposal OECD 488 submitted

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:

other: Acceptable publication which meets basic scientific principles

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 - 14 weeks

Route of administration:

intraperitoneal

Vehicle:

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

Duration of treatment / exposure:

30 h

Frequency of treatment:

once

Post exposure period:

30 h

Remarks:

Doses / Concentrations:
345, 603, 861 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

4 animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

no data

Tissues and cell types examined:

polychromatic erythrocytes of bone marrow

Details of tissue and slide preparation:

The smears were stained according to the method of Schmid (Schmid W. 1976; Chemical Mutagens; edited by A . Hollaender; Vol4; p31; Plenum Press, New York )
Slides were scored as described by Wild (Wild D. 1980; Archs Toxicol.; 43; 249).

Statistics:

Statistical significance was determined according to the methods of Kastenbaum & Bowman (Kastenbaum M. 1970; Mutation Res. 9; 527)

Sex:

female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Results expressed as mean number of micronucleated polychromatic erythrocytes (PE) per 1000 PE:

345 mg/kg bw: 1.5 (4/4 animals survived)

603 mg/kg bw: 1.0 (4/4 animals survived)

861 mg/kg bw: 2.0 (4/4 animals survived)

vehicle control: 1.5 (4/4 animals survived)

mean of the vehicle controls for all substances tested: 1.85

A structurally and metabolically related substance (Hydroxycitronellol CAS. 107-74-4)

was tested under similar testing conditions:

516 mg/kg bw: 2.2 (4/4 animals survived)

860 mg/kg bw: 2.2 (4/4 animals survived)

1204 mg/kg bw: 2.6 (3/7 animals survived)

vehicle control: 2.0 (4/4 animals survived)

mean of the vehicle controls for all substances tested: 1.85

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Mutagenicity in bacteria
In the chosen key study, i.e. an Ames test reported in literature similar to OECD TG 471, bacteria S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538 were treated with concentrations up to 3.6 mg/plate hydroxycitronellal (no info on vehicle used) with and without metabolic activation by Aroclor 1254-induced rat liver S9 mix (Wild 1983). Hydroxycitronellal was reported to be negative under the chosen testing conditions and is therefore to be considered as non-mutagenic in bacteria.


Mutagenicity in mammalian cells in vitro
In the chosen key study, i.e. an HPRT tests according to OECD TG 476 and GLP, hydroxycitronellal is considered to induce forward mutations in the HPRT locus assay under in vitro conditions in CHO cells in the absence of metabolic activation.

A testing porposal has been submitted as a mammalian erythrocyte Pig-a gene mutation (OECD TG 470) should be conducted to clarify the ambiguous and insufficient information on mutagenicity for the test substance.

 

 

Cytogenicity/mutagenicity in mammals in vivo

In the chosen key study, i.e. a micronucleus test reported in literature, four NMRI mice were treated intraperitoneally with concentrations of 345, 603, 861 mg/kg bw. After 30 hours, polychromatic erythrocytes of the bone marrow were analyzed for the presence of micronuclei. The mean ratio of micronucleated PE/1000 PE ranged from 1.0 - 2.0 and was comparable to the ratio observed in vehicle treated animals (1.5 PE/1000 PE). Overall, hydroxycitronellal was found to be not cytogenic in mice. In support, the same publication reported the metabolically related alcohol, i.e. hydroxycitronellol (CAS. 107-74-4), to be negative under similar testing conditions

In a Drosophila SLRL test not according to current standard protocols, incubation with hydroxycitronellal (and the metabolically related alcohol hydroxycitronellol) did not lead to an evident increase in the numbers of sex-linked recessive lethal mutations (Wild 1983).

Short description of key information:
Genetic toxicity:
- Mutagenicity in bacteria in vitro/ Ames: negative (Wild 1983)
- Mutagenicity in mammalian cells in vitro/ HPRT: positive (BASF 2018)
- Cytogenicity in mammals in vivo/ MNT: negative (Wild 1983)

Endpoint Conclusion:
The present data on genetic toxicity are ambiguous and thus, a testing proposal for an additional in vivo study according to OECD TG 470 has been submitted.

Justification for classification or non-classification

The present data on genetic toxicity are ambiguous and thus, a testing proposal for an additional in vivo study according to OECD 470 has been submitted. A definite statement on classification of the test substance concerning genotoxicity according to criteria laid down in Regulation (EC) 1272/2008 has to be postponed until results of the mammalian erythrocyte Pig-a gene mutation assay will be available.