4-hydroxybenzaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not increase the number of revertant colony numbers in the bacterial reverse mutation assay according to OECD 471. Thus, the test item is not considered to be mutagenic in bacteria (reference 7.6.1 -1).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May - 29 Jun 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his and trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
Phenobarbital/-naphthoflavone induced rat liver S9 was used as the metabolic activation system.
- method of preparation of S9 mix
The S9 is prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin; 22335 Hamburg, Germany) and by peroral administrations of -naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week.
- concentration or volume of S9 mix and S9 in the final culture medium
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix.
- quality controls of S9
Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation, TA 1535, TA 100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Without metabolic activation, TA 1537, TA 98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation, WP2 uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation, TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) and preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 1 hour at 37 °C
- Exposure duration/duration of treatment: 48 hours at 37 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. Since no toxic effects were observed 5000 µg/plate were chosen as maximal concentration.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
see attached results

- Signs of toxicity
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed with and without metabolic activation in experiment I. In experiment II, toxic effects were observed only in strain WP2 uvrA with and without
metabolic activation at 5000 µg/plate.

- Mean number of revertant colonies per plate and standard deviation
see attached results

- Genotoxicity results:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

HISTORICAL CONTROL DATA
- see table 1

Table 1 HISTORICAL CONTROL DATA

These data represent the laboratory's historical control data from January 2011 until December 2011 representing approx. 550 experiments (WP2 uvrA the historical data are based on approx. 200 experiments).

Strain		without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control	14	2.37	9	23	20	3.75	11	35
	Untreated control	14	2.87	7	25	20	3.82	10	32
	Positive control	1751	226.44	710	2385	367	93.12	126	703
TA1537	Solvent control	12	3.31	5	26	16	4.34	7	30
	Untreated control	12	4.03	5	29	18	4.92	6	33
	Positive control	88	38.92	51	448	342	144.31	77	809
TA98	Solvent control	30	5.60	17	47	40	6.08	21	58
	Untreated control	31	6.36	17	55	42	6.83	24	68
	Positive control	372	78.05	158	595	2167	717.60	249	4089
TA 100	Solvent control	142	29.42	86	243	156	29.4	99	249
	Untreated control	150	28.19	86	248	163	31.26	94	281
	Positive control	1741	488.75	569	3082	2642	796.59	825	4503
WP2/uvA	Solvent control	49	7.67	32	69	60	8.55	31	86
	Untreated control	50	7.64	31	71	60	8.06	38	83
	Positive control	697	336.39	174	1435	368	108.91	176	1534

Mean = mean value of revertants/plate

SD = standard deviation Min = minimal value/Max = maximal value

 

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

This study according OECD TG 471 was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed with and without metabolic activation in experiment I. In experiment II, toxic effects were observed only in strain WP2 uvrA with and without metabolic activation at 5000 µg/plate. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 4the substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

This study according OECD TG 471 was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed with and without metabolic activation in experiment I. In experiment II, toxic effects were observed only in strain WP2 uvrA with and without metabolic activation at 5000 µg/plate. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 4the substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for fourteenth time in Regulation (EU) No 2020/217.

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