Benzaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Benzaldehyde was tested in contemporary OECD TG 487 (2016) and TG 490 (2016) compliant studies. An OECD 487 (2016) in vitro micronucleus study in human lymphocytes was conducted up to toxic concentrations for 3 hours in the absence and presence of rat liver metabolic activation system (S9) and for 24 hours in the absence of S9. Under the experimental conditions, it was concluded that benzaldehyde did not induce biologically relevant increases in the frequency of micronuclei.

The OECD 490 (2016) in vitro mammalian cell gene mutation test was conducted up to precipitating concentrations. Under the experimental conditions, benzaldehyde did not induce mutation at the tk locus of mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence or presence of a rat liver metabolic activation system (S9).

A number of historical ames studies published in the scientific literature all resulted in a negative response both with and without metabolic activation.

With the completion of contemporary OECD TG 487 and TG 490 studies, the overall Weight of Evidence supports the classification of benzaldehyde as "not genotoxic". The whole of the literature points to benzaldehyde as not genotoxic, with only a small number of studies concluding "positive" results. In particular the following citations however these studies are not compliant with current OECD test guidelines with methodological deviations identified.

Citation	Test System	Conclusion
Kasamaki et al (1982)	Chinese hamster cell line CH-B-241	Equivocal
Sofuni et al (1985)	Chinese hamster cells	Positive in the absence of S9
Jansson et al (1988)	Primary lymphocytes obtained from healthy non-smoking donors	Positive
Heck et al (1989)	Tk +/- 3.7.2C heterozygote of L5178Y mouse lymphoma cell line	Equivocal
McGregor et al (1991)	Tk +/- 3.7.2C heterozygote of L5178Y mouse lymphoma cell line	Positive without S9, increases in mutant fraction were "very close to highly toxic doses"

Several in vitro and in vivo genotoxicity tests with the test substance are available. The NTP report on the test substance (NTP, 1990) concluded that benzaldehyde was not mutagenic in six strains of S. typhimurium and did not induce chromosomal aberrations in CHO cells, with or without exogenous metabolic activation. Benzaldehyde induced increases in trifluorothymidine-resistant mouse lymphoma cells in the absence of exogenous metabolic activation (McGregor et al, 1991) and increased sister chromatid exchanges in CHO cells in both the presence and absence of metabolic activation. Sex-linked recessive lethal mutations were not induced in the germ cells of adult male D. melanogaster administered benzaldehyde by feeding or by injection. However taking into consideration the key studies from the contemporary OECD 487 and 490 compliant GLP studies with the historical studies (with some methodological short comings), it is concluded that benzaldehyde is considered not to be genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2018 - 17 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Kalama Chemical BV (The Netherlands); Lot No. 1803-1
- Expiration date of the lot/batch: 2020-01-15
- Purity test date: 2018-09-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at 15-25°C, protected from light under nitrogen
- Stability under test conditions: no analyses of the stability of the test article in administered formulations or dilutions was undertaken as fresh preparations of test article were employed
- Solubility and stability of the test substance in the solvent/vehicle: Preliminary solubility data indicated that Kalama® Benzaldehyde FCC grade was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 119.5 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test article stock solutions were prepared by formulating Kalama® Benzaldehyde FCC grade under subdued lighting in DMSO, with the aid of vortex mixing, to give
the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 4 hours of initial formulation.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : colourless liquid

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood from two healthy, non-smoking Female volunteers from a panel of donors at Covance was used for each experiment.
- Suitability of cells: The use of human peripheral blood lymphocytes is recommended because the cells are only used in short-term culture and maintain a stable karyotype (Evans & O’Riordan, 1975).
- Cell cycle length, doubling time or proliferation index: Cell cycle length: 13 ± 2 hour
- Sex, age and number of blood donors if applicable: Blood from two healthy, non-smoking Female volunteers (Range-Finder: 34, 30 years; Micronucleus Experiment: 23, 34 Years)
- Whether whole blood or separated lymphocytes were used if applicable: human lymphocyte cultures prepared from the pooled blood of two female donors

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S9) prepared from male Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Cytotoxicity Range-Finder Experiment:
3+21 Hour Treatment (-S9): 3.853, 6.422, 10.70, 17.84, 29.73, 49.55, 82.58, 137.6, 229.4, 382.3, 637.2, 1062 µg/mL
3+21 Hour Treatment (+S9): 3.853, 6.422, 10.70, 17.84, 29.73, 49.55, 82.58, 137.6, 229.4, 382.3, 637.2, 1062 µg/mL
24+24 Hour Treatments (-S9): 3.853, 6.422, 10.70, 17.84, 29.73, 49.55, 82.58, 137.6, 229.4, 382.3, 637.2, 1062 µg/mL

A maximum concentration of 1062 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a maximum concentration equivalent to 10 mM (a suitable maximum concentration for in vitro genetic toxicology assays of this type), based on the test article molecular weight of 106.121. No marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder (1062 µg/mL), compared to the concurrent vehicle controls. The results of the cytotoxicity Range-Finder Experiment were used to select suitable maximum concentrations for the Micronucleus Experiment.

Micronucleus Experiment:
3+21 Hour Treatments (-S9): 100, 200, 400, 500, 600, 650, 700, 750, 800, 850, 950, 1062 µg/mL
3+21 Hour Treatments (+S9): 100, 200, 400, 500, 600, 650, 700, 750, 800, 850, 950, 1062 µg/mL
24+24 Hour Treatments (-S9): 50, 100, 150, 200, 250, 300, 350, 400, 450, 600 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Kalama® Benzaldehyde FCC grade was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 119.5 mg/mL.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Vinblastine (VIN): Treatment Regime: -S9: 24+24 (concentration: 0.04 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: approximately 48 hours
- Exposure duration: 3 (±S9) or 24 hours (-S9)
- Fixation time (start of exposure up to fixation or harvest of cells): -S9 (3 hour treatment): 72 hours; -S9 (24 hour treatment): 96 hours; + S9 (3 hour treatment): 72 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (Cyto-B (formulated in DMSO) added to post wash-off culture medium to give a final concentration of 6 µg/mL per culture.

STAIN (for cytogenetic assays): Acridine Orange in phosphate buffered saline (PBS), pH 6.8

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, two to three minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried and stored protected from light at room temperature prior to staining. Slides were stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes and washed with PBS (with agitation) for a few seconds. The quality of the staining was checked. Slides were air-dried and stored protected from light at room temperature. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.

NUMBER OF CELLS EVALUATED: A minimum ofone thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. For the 24 hour treatment in the absence of S9, an additional 1000 binucleate cells from each culture (therefore 4000 per concentration) were analysed from the test article concentrations selected for analysis.


CRITERIA FOR MICRONUCLEUS IDENTIFICATION: A micronucleus was only recorded if it met the following criteria:

1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Relative index (RI)
- Any supplementary information relevant to cytotoxicity: Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined. RI, which indicates the relative number of nuclei compared to vehicle controls was determined using the formula as follows:

RI = (number binucleate cells + 2 (number multinucleate cells) / total number of cells in treated cultures)

Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows:

Relative RI (%) = (RI of treated cultures / RI of vehicle controls) x 100

Cytotoxicity (%) is expressed as (100 – Relative RI)

A selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.

Rationale for test conditions:

Please see 'Any other information on materials and methods incl. tables' for information on Rationale for Test Conditions.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:

1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed

2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed

3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).

The test article was considered positive in this assay if all of the above criteria were met.

The test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations (Thybaud et al., 2007).

Statistics:

After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.

The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989).

The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p ≤0.05 were accepted as significant.

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder (1062 µg/mL), compared to the concurrent vehicle controls (individual data not reported).

The proportion of micronucleated binucleate (MNBN) cells in negative (vehicle) control cultures fell within (or very close to) the 95th percentile of the current observed historical vehicle control (normal) ranges. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.

All acceptance criteria were considered met and the study was accepted as valid.


Validity of Study
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures for the 3+21 hour and 24+24 hour treatments in the absence of S9. Statistically significant heterogeneity (p≤0.01) was observed for the 3+21 hour treatment in the presence of S9, primarily due to large differences in MNBN cell frequencies between replicates for the vehicle control and the lowest test article concentration analysed (400 µg/mL). However, none of the MNBN cell frequency values exceeded the normal range under this treatment condition, therefore the observed heterogeneity did not affect the interpretation of the data

2. The frequency of MNBN cells in vehicle controls fell within the normal ranges with the exception of two of the four vehicle control cultures analysed for the 24+24 hour treatment in the absence of S9, one of which fell within the observed range. However, the mean vehicle control MNBN cell frequency was within the normal range and the data were considered acceptable.

3. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range.

4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest.

5. The maximum concentration analysed under each treatment condition met the criteria specified.

Table 4. Range-Finder: -S9: Results of the 3+21 Hour Treatments
Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	64	125	11	200	0.74
	B	52	133	15	200	0.82
	Total	116	258	26	400	0.78	-
3.853	A	NSc	-	-	-	-	-
6.422	A	NSc	-	-	-	-	-
10.70	A	NSc	-	-	-	-	-
17.84	A	NSc	-	-	-	-	-
29.73	A	42	148	10	200	0.84	0
49.55	A	58	139	3	200	0.73	6
82.58	A	49	140	11	200	0.81	0
137.6	A	54	135	11	200	0.79	0
229.4	A	52	138	10	200	0.79	0
382.3	A	75	123	2	200	0.64	18
637.2	A	96	104	0	200	0.52	33P
1062	A	183	17	0	200	0.09	89P

P = Precipitation observed at treatment

NSc = Not scored

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

 

Table 5. Range-Finder: +S9: Results of the 3+21 Hour Treatments
Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	50	139	11	200	0.81
	B	56	127	17	200	0.81
	Total	106	266	28	400	0.81	-
3.853	A	NSc	-	-	-	-	-
6.422	A	NSc	-	-	-	-	-
10.70	A	NSc	-	-	-	-	-
17.84	A	64	122	14	200	0.75	7
29.73	A	55	133	12	200	0.79	2
49.55	A	58	137	5	200	0.74	9
82.58	A	45	138	17	200	0.86	0
137.6	A	59	132	9	200	0.75	7
229.4	A	57	138	5	200	0.74	8
382.3	A	78	118	4	200	0.63	22
637.2	A	96	102	2	200	0.53	34P
1062	A	177	23	0	200	0.12	86P

P = Precipitation observed at treatment

NSc = Not scored

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

 

Table 6. Range-Finder: +S9: Results of the 24+24 Hour Treatments
Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	28	146	26	200	0.99
	B	39	142	19	200	0.90
	Total	67	288	45	400	0.95	-
3.853	A	NSc	-	-	-	-	-
6.422	A	NSc	-	-	-	-	-
10.70	A	NSc	-	-	-	-	-
17.84	A	31	148	21	200	0.95	0
29.73	A	24	152	24	200	1.00	0
49.55	A	23	155	22	200	1.00	0
82.58	A	48	144	8	200	0.80	15
137.6	A	80	118	2	200	0.61	35
229.4	A	124	74	2	200	0.39	59
382.3	A	119	74	7	200	0.55	53
637.2	A	194	6	0	200	0.03	97P
1062	A	199	1	0	200	0.01	99P

P = Precipitation observed at treatment

NSc = Not scored

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

Table 7. Micronucleus Experiment: -S9: Results of the 3+21 Hour Treatments
Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	112	360	28	500	0.83
	B	127	347	26	500	0.80
	C	85	380	35	500	0.90
	D	88	378	34	500	0.89
	Total	412	1465	123	2000	0.86	-
100.0	A	134	344	22	500	0.78
	B	114	365	21	500	0.81
	Total	248	709	43	1000	0.80	7
200.0	A	143	328	29	500	0.77
	B	160	315	25	500	0.73
	Total	303	643	54	1000	0.75	12
400.0	A	140	348	12	500	0.74
	B	152	337	11	500	0.72
	Total	292	685	23	1000	0.73	15 #
500.0	A	183	311	6	500	0.65
	B	170	325	5	500	0.67
	Total	353	636	11	1000	0.66	23
600.0	A	213	286	1	500	0.58
	B	244	255	1	500	0.51
	Total	457	541	2	1000	0.55	36 #
650.0	A	246	249	5	500	0.52
	B	237	261	2	500	0.53
	Total	483	510	7	1000	0.52	39
700.0	A	225	271	4	500	0.56
	B	279	219	2	500	0.45
	Total	504	490	6	1000	0.50	41
750.0	A	265	234	1	500	0.47
	B	285	213	2	500	0.43
	Total	550	447	3	1000	0.45	47
800.0	A	305	193	2	500	0.39
	B	294	206	0	500	0.41
	Total	599	399	2	1000	0.40	53 #
850.0	A	308	191	1	500	0.39
	B	303	194	3	500	0.40
	Total	611	385	4	1000	0.39	54
950.0	A	348	151	1	500	0.31
	B	381	117	2	500	0.24
	Total	729	268	3	1000	0.27	68 P
1062	A	473	27	0	500	0.05
	B	456	44	0	500	0.09
	Total	929	71	0	1000	0.07	92 P
MMC, 0.30	A	250	249	1	500	0.50
	B	244	255	1	500	0.51
	Total	494	504	2	1000	0.51	41 #

P = Precipitation observed at treatment

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

# Highlighted concentrations selected for analysis

 

Table 8. Micronucleus Experiment: +S9: Results of the 3+21 Hour Treatments
Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	132	341	27	500	0.79
	B	117	342	41	500	0.85
	C	115	369	16	500	0.80
	D	110	366	24	500	0.83
	Total	474	1418	108	2000	0.82	-
100.0	A	144	343	13	500	0.74
	B	106	366	28	500	0.84
	Total	250	709	41	1000	0.79	3
200.0	A	124	364	12	500	0.78
	B	103	379	18	500	0.83
	Total	227	743	30	1000	0.80	2
400.0	A	148	343	9	500	0.72
	B	130	363	7	500	0.75
	Total	278	706	16	1000	0.74	10 #
500.0	A	204	292	4	500	0.60
	B	187	310	3	500	0.63
	Total	391	602	7	1000	0.62	25
600.0	A	250	247	3	500	0.51
	B	239	259	2	500	0.53
	Total	489	506	5	1000	0.52	37 #
650.0	A	258	237	5	500	0.49
	B	255	244	1	500	0.49
	Total	513	481	6	1000	0.49	40
700.0	A	230	269	1	500	0.54
	B	274	226	0	500	0.45
	Total	504	495	1	1000	0.50	39
750.0	A	270	227	3	500	0.47
	B	270	230	0	500	0.46
	Total	540	457	3	1000	0.46	43
800.0	A	321	177	2	500	0.36
	B	303	197	0	500	0.39
	Total	624	374	2	1000	0.38	54 #
850.0	A	353	145	2	500	0.30
	B	317	181	2	500	0.37
	Total	670	326	4	1000	0.33	59
950.0	A	370	128	2	500	0.26
	B	366	134	0	500	0.27
	Total	736	262	2	1000	0.27	67 P
1062	A	415	84	1	500	0.17
	B	403	96	1	500	0.20
	Total	818	180	2	1000	0.18	77 P
CPA, 3.00	A	181	312	7	500	0.65
	B	151	342	7	500	0.71
	Total	332	654	14	1000	0.68	17
CPA, 5.00	A	218	280	2	500	0.57
	B	235	264	1	500	0.53
	Total	453	544	3	1000	0.55
CPA, 7.00	A	244	255	1	500	0.51
	B	243	257	0	500	0.51
	Total	487	512	1	1000	0.51	37 #

P = Precipitation observed at treatment

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

# Highlighted concentrations selected for analysis

 

Table 9. Micronucleus Experiment: -S9: Results of the 24+24 Hour Treatments
Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	33	347	120	500	1.17
	B	41	326	133	500	1.18
	C	38	328	134	500	1.19
	D	32	366	102	500	1.14
	Total	144	1367	489	2000	1.17
50.0	A	46	353	101	500	1.11
	B	53	342	105	500	1.10
	Total	99	695	206	1000	1.11	6 #
100.0	A	113	363	24	500	0.82
	B	124	346	30	500	0.81
	Total	237	709	54	1000	0.82	30 #
150.0	A	151	331	18	500	0.73
	B	174	320	6	500	0.66
	Total	325	651	24	1000	0.70	40
200.0	A	264	230	6	500	0.48
	B	222	269	9	500	0.57
	Total	486	499	15	1000	0.53	55 #
250.0	A	337	158	5	500	0.34
	B	266	222	12	500	0.49
	Total	603	380	17	1000	0.41	65
300.0	A	285	188	27	500	0.65
	B	312	175	13	500	0.40
	Total	697	363	40	1000	0.44	62
350.0	A	212	250	38	500	0.65
	B	262	202	36	500	0.55
	Total	474	452	74	1000	0.60	49
400.0	A	275	211	14	500	0.48
	B	291	199	10	500	0.44
	Total	566	410	24	1000	0.46	61
450.0	A	348	147	5	500	0.31
	B	363	134	3	500	0.28
	Total	711	281	8	1000	0.30	75
600.0	A	478	21	1	500	0.05
	B	478	22	0	500	0.04
	Total	956	43	1	1000	0.05	96
VIN, 0.04	A	195	225	80	500	0.77
	B	227	199	74	500	0.69
	Total	422	424	154	1000	0.73	38 #

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

# Highlighted concentrations selected for analysis

 

Table 10. Summary of Results of the Micronucleus Test
Treatment	Concentration (µg/mL)	Cytotoxicity (%)^$	Mean MNBN Cell Frequency (%)	Historical Control Range (%)^#	Statistical Significance
3+21 hour -S9	Vehicle^a	-	0.60	0.00 to 1.01	-
	400.0	15	0.60		NS
	600.0	36	0.45		NS
	800.0	53	0.50		NS
	*MMC, 0.30	41	7.00		p≤0.001
3+21 hour +S9	Vehicle^a	-	0.50	0.10 to 1.20	-
	400.0	10	0.35		NS
	600.0	37	0.90		NS
	800.0	54	0.80		NS
	*CPA, 7.00	37	3.10		p≤0.001
24+24 hour -S9	Vehicle^a	-	0.75	0.10 to 0.80	-
	50.0	6	0.60		NS
	100.0	30	0.40		NS
	200.0	55	0.88		NS
	*VIN 0.04	38	2.95		p≤0.001

^a Vehicle control was DMSO

* Positive control

^# 95^thpercentile of the observed range

^$ Based on replication index

NS Not significant

Conclusions:

The test material (Kalama® Benzaldehyde FCC grade) did not induce biologically relevant increases in the frequency of micronuclei when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S9) and for 24+24 hours in the absence of S9 under the experimental conditions described.

Executive summary:

In a key OECD Guideline 487 study, the test material (Kalama® Benzaldehyde FCC grade) was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S9) from Aroclor 1254-induced rats.

 

The test material was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). The highest concentrations analysed in the Micronucleus Experiment were limited by cytotoxicity under each treatment condition and were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test material concentrations for micronucleus analysis were selected by evaluating the effect of Kalama® Benzaldehyde FCC grade on the replication index (RI). Micronuclei were analysed at three concentrations.

 

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in these cultures fell within (or very close to) the 95^thpercentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.

 

All acceptance criteria were considered met and the study was accepted as valid.

 

Treatment of cells with the test material for 3+21 hours in the absence and presence of S9and for 24+24 hours in the absence of S9 resulted in frequencies of MNBN cells that were generally similar to and not significantly different (at the p≤0.05 level), compared to those observed in the concurrent vehicle controls, at any concentration analysed under each treatment condition. The MNBN cell frequencies fell within the normal ranges at all concentrations analysed with the exception of one culture at the highest concentration analysed following the 24+24 hour treatment in the absence of S9 (1% at 200 µg/mL, which gave 55% mean cytotoxicity). However, there were no statistically significant increases in MNBN cell frequency at any concentration analysed following the 24+24 hour treatment in the absence of S9 and no statistically significant linear trend, therefore this isolated observation was considered not biologically relevant. A statistically significant linear trend (p≤0.05) was observed following the 3+21 hour treatment in the presence of S9 but as there were no statistically significant increases in MNBN cell frequency at any concentration analysed, this observation was also considered not biologically relevant.

 

It was concluded that the test material (Kalama® Benzaldehyde FCC grade) did not induce biologically relevant increases in the frequency of micronuclei when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S9) and for 24+24 hours in the absence of S9 under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-25 to 2019-12-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Kalama Chemical BV (The Netherlands); Lot #: 1803-1
- Expiration date of the lot/batch: 2020-01-15
- Purity test date: 2018-09-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at 15-25°C, protected from light under nitrogen
- Stability under test conditions: no analyses of the stability of the test article in administered formulations or dilutions was undertaken as fresh preparations of test article were employed
- Solubility and stability of the test substance in the solvent/vehicle: Preliminary solubility data indicated that Kalama® Benzaldehyde FCC grade was miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 119.5 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test article stock solutions were prepared by formulating Kalama® Benzaldehyde FCC grade under subdued lighting in DMSO, with the aid of vortex mixing, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 3.5 hours of initial formulation.

FORM AS APPLIED IN THE TEST (if different from that of starting material): colourless liquid

Target gene:

tk (thymidine kinase) locus in mouse lymphoma L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance
- Suitability of cells: The tk (thymidine kinase) locus in mouse lymphoma L5178Y cells is capable of detecting both gene mutations and chromosome aberrations.
- Methods for maintenance in cell culture if applicable: The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37±1°C. When the cells were growing well, subcultures were established in an appropriate number of flasks.

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free

Metabolic activation:

with and without

Metabolic activation system:

Obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Cytotoxicity Range-Finder Experiment (±S9): 0, 33.19, 66.38, 132.8, 265.5, 531, and 1062 µg/mL

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S9, ranging from 33.19 to 1062 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide greater than 10% relative suspension growth (RSG) was 531 µg/mL, which gave 41% and 32% RSG in the absence and presence of S9, respectively.

In the Mutation Experiment ten concentrations, ranging from 100 to 1062 µg/mL, were tested in the absence and presence of S9. Two days after treatment the highest concentration analysed to determine viability and TFT resistance was 800 µg/mL, which gave 16% and 17% relative total growth (RTG) in the absence and presence of S9, respectively.

Mutation Experiment (±S9): 0, 100, 200, 400, 500, 600, 700, and 800 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Kalama® Benzaldehyde FCC grade was miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 119.5 mg/mL.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): At least 10^7 cells in a volume of 17.8 mL tissue culture medium (cells in RPMI 10 diluted with RPMI A to give a final concentration of 5% serum) were used.

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 2 days:

NUMBER OF REPLICATIONS: Each treatment, in the absence or presence of S9, was in duplicate (single cultures only used for positive control treatments)

NUMBER OF CELLS EVALUATED:
Plating for Viability – Mutation Experiment:
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10^4 cells/mL in readiness for plating for TFT resistance. Samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well).

Plating for TFT Resistance - Mutation Experiment:
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10^4 cells/mL. TFT (300 µg/mL) was diluted approximately 100 fold into these suspensions to give a final concentration of 3 µg/mL. Using an eight-channel pipette, 0.2 mL of each suspension was placed into each well of four 96-well microtitre plates (384 wells at 2 x 10^3 cells/well).

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth
- Any supplementary information relevant to cytotoxicity: Relative Suspension Growth (RSG) is a measure of the growth in suspension during treatment and the expression period relative to the mean control.

Rationale for test conditions:

Preliminary solubility data indicated that Kalama® Benzaldehyde FCC grade was miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 119.5 mg/mL. The solubility limit in culture medium was in excess of 1195 µg/mL, as indicated by the absence of precipitate at this concentration 24 hours after test article addition, with warming at 37°C. A maximum concentration of 1062 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a maximum concentration equivalent to 10 mM (a suitable maximum concentration for in vitro genetic toxicology assays of this type), based on the test article molecular weight of 106.121. Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:

1. The MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF

2. The linear trend test was statistically significant.

The test article was considered positive in this assay if both of the above criteria were met.

The test article was considered negative in this assay if neither of the above criteria were met.

Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Please see 'Any other information on materials and methods incl. tables' for information on statistics

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity Range-Finder Experiment:
Six concentrations were tested in the absence and presence of S9, ranging from 33.19 to 1062 µg/mL (equivalent to 10 mM at the highest concentration tested). Upon addition of the test material to the cultures, precipitate was observed at the highest two concentrations tested in the absence and presence of S9 (531 and 1062 µg/mL) but no post-treatment precipitate was observed under either treatment condition. The highest concentration to provide greater than 10% RSG was 531 µg/mL, which gave 41% and 32% RSG in the absence and presence of S9, respectively (Table 1).

No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (1062 µg/mL), compared to the concurrent vehicle controls.

Mutation Experiment:
Ten concentrations, ranging from 100 to 1062 µg/mL, were tested in the absence and presence of S9. Upon addition of the test article to the cultures, precipitate was observed at the highest five concentrations tested in the absence and presence of S9 (700 to 1062 µg/mL) but no post-treatment precipitate was observed under either treatment condition. Two days after treatment the highest three concentrations tested in the absence of S9 (900 to 1062 µg/mL) and the highest concentration tested in the presence of S9 (1062 µg/mL) were considered too toxic for selection to determine viability and TFT resistance. All other concentrations in the absence and presence of S9 were selected. However, the highest two concentrations selected in the presence of S9 (900 and 1000 µg/mL) were later rejected from analysis due to extreme toxicity (<10% RTG). The highest concentration analysed was 800 µg/mL, which gave 16% and 17% RTG in the absence and presence of S9, respectively (Table 2).

The acceptance criteria were met, with one exception. The mean vehicle control MF value in the presence of S9 was 46.53 mutants per 106 viable cells, which fell slightly below the acceptable range of 50 to 170 mutants per 106 viable cells. However, there were clearly no notable increases in MF (above the GEF) in any treated culture in the presence of S9 and the positive control chemical B[a]P showed clear induction of mutation, therefore the data were considered acceptable and valid.

When tested up to toxic concentrations in the Mutation Experiment, the MF values of the concentrations plated were all less than the sum of the mean control MF plus the GEF, indicating a negative result. A statistically significant linear trend was observed in the presence of S9, but in the absence of any increases in MF which exceeded the GEF in any treated culture under this treatment condition, this observation was considered not to be biologically relevant.

In addition, for the negative and positive controls, the number of wells containing small colonies and the number containing large colonies were scored. Thus the small and large colony MF could be estimated and the proportion of small mutant colonies could be calculated. For the vehicle controls, the proportion of small colony mutants in the absence and presence of S9 ranged from 38% to 42%. Marked increases in the number of both small and large colony mutants were observed following treatment with the positive control chemicals MMS and B[a]P.

Table 1: RSG Values - Range-Finder Experiment
Concentration (µg/mL)	3 Hour Treatment (-S9) % RSG	3 Hour Treatment (+S9) % RSG
0	100	100
33.19	98	65
66.38	95	100
132.8	91	77
265.5	87	65
531 P	41	32
1062 P	2	3

% RSG: Percent relative suspension growth

P: Precipitation observed at the time of treatment

Table 2: Summary of Mutation Data
3 Hour Treatment -S9			3 Hour Treatment +S9
Concentration µg/mL	%RTG	MF §	Concentration µg/mL	%RTG	MF §
0	100	52.67	0	100	46.53
100	73	52.90	100	69	66.75
200	52	85.52	200	63	74.33
400	44	68.00	400	48	78.03
500	29	88.62	500	38	72.73
600	28	75.80	600	33	82.08
700 P	16	50.53	700 P	21	111.11
800 P	16	58.64	800 P	17	99.83
MMS 15	48	316.78	B[a]P 2	35	575.56
MMS 20	42	370.08	B[a]P 3	30	640.12

Linear trend test on mutant frequency in the absence of S9: p-value = 0.4554, not significant

Linear trend test on mutant frequency in the presence of S9: p-value = 0.001 (** P < 0.01)

 

§: 5-TFT-resistant mutants/10⁶ viable cells 2 days after treatment

% RTG: Percent Relative Total Growth

P: Precipitation noted at time of treatment

Conclusions:

The test material (Kalama® Benzaldehyde FCC grade) did not induce mutation at the tk locus of mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S9) under the experimental conditions described.

Executive summary:

In a key OECD Guideline 490 in vitro gene mutation study, the test material (Kalama® Benzaldehyde FCC grade)was assayed for the ability to induce mutation at the tk locus (5‑trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S9). The test material was formulated inanhydrous analytical grade dimethyl sulphoxide(DMSO).

 

A 3 hour treatment incubation period was used for each experiment.

 

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S9, ranging from 33.19 to 1062 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide greater than 10% relative suspension growth (RSG) was 531 µg/mL, which gave 41% and 32% RSG in the absence and presence of S9, respectively.

 

In the Mutation Experiment ten concentrations, ranging from 100 to 1062 µg/mL, were tested in the absence and presence of S9. Two days after treatment the highest concentrationanalysed to determine viability and TFT resistance was 800 µg/mL, which gave 16% and 17% relative total growth (RTG) in the absence and presence of S9, respectively.

 

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S9. Mutant frequencies (MF) in vehicle control cultures fell within (or very close to) acceptable ranges. Clear increases in mutation were induced by the positive control chemicals, Methyl methane sulphonate (without S9) and Benzo[a]pyrene (with S9). Therefore the study was accepted as valid.

 

When tested up to toxic concentrations in the Mutation Experiment, the MF values of the concentrations plated were all less than the sum of the mean control MF plus the Global Evaluation Factor (GEF, 126 mutants per 10⁶viable cells), indicating a negative result.A statistically significant linear trend was observed in the presence of S9, but in the absence of any increases in MF which exceeded the GEF in any treated culture under this treatment condition, this observation was considered not biologically relevant.

 

Based on the results observed, the study authors concluded that the test material (Kalama® Benzaldehyde FCC grade) did not induce mutation at the tk locus of mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S9) under the experimental conditions described.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1978 - 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Regarding the requirements under Annex X, Section 8.4.1 "In vitro gene mutation study in bacteria", while there is not a single study performed exactly to guideline, there are numerous studies which collectively cover all of the strains and conditions set forth in the current OECD 471.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Dillon et al. (1998)

Deviations:

yes

Remarks:

Did not evaluate tester strains TA1535 or TA98.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Florin et al. (1980)

Deviations:

yes

Remarks:

Did not evaluate E. coli WP2 uvrA/E. coli WP2 uvrA (pKM101) or S. Typhimurium TA102

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Fujita et al. (1992)

Deviations:

yes

Remarks:

This study only evaluted TA97 and TA102.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Haworth et al. (1983)

Deviations:

yes

Remarks:

This study did not evaluate E. coli WP2 uvrA/E. coli WP2 uvrA (pKM101)/TA 102

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Heck et al. (1989)

Deviations:

yes

Remarks:

This study did not evaluate E. coli WP2 uvrA/E. coli WP2 uvrA (pKM101)/TA 102. In addition, there is no mention of positive controls.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Kasamaki et al. (1982)

Deviations:

yes

Remarks:

This study only evaluates TA 98 and TA100.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Nohmi et al. (1985)

Deviations:

yes

Remarks:

Evaluated only two common strains of S. typhumurium (TA98 and TA100), and one non-standard (TA2637).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Sasaki et al. (1978)

Deviations:

yes

Remarks:

Only strains TA98 and TA100 were evaluated. Only abstract available, with no mention of positive controls.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Yamada and Honma (2018) summarizes previous studies.

Principles of method if other than guideline:

- Principle of test:
- Short description of test conditions:
- Parameters analysed / observed:

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

Dillon et al. (1998) – Obtained from Radian Corporation (Austin, TX), but original supplier listed as LaPine Scientific. Stated as “72 – 91% purity”. No expiration date mentioned.
Florin et al. (1980) – Source/purity/Lot No./Expiration Date not specified.
Fujita et al. (1992) – Wako Pure Chemicals, purity/Lot No./expiration date not specified.
Haworth et al. (1983) – Obtained from Radian Corporation (Austin, TX), but original supplier listed as LaPine Scientific. Stated as “72 – 91% purity”. Lot No. 45,519, expiration date not specified.
Heck et al. (1989) – Source/purity/Lot No./Expiration Date not specified.
Kasamaki et al. (1982) – Obtained from Nakarai Pharmaceutical Company (Japan). Purity/Lot No./Expiration Date not specified.
Nohmi et al. (1985) – Source/purity/Lot No./Expiration Date not specified.
Sasaki and Endo (1978) – Source/purity/Lot No./Expiration Date not specified.

Species / strain / cell type:

S. typhimurium, other: TA 100, TA 102 and TA 104

Remarks:

Dillon et al. (1998)

Species / strain / cell type:

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

Remarks:

Florin et al. (1980)

Species / strain / cell type:

S. typhimurium, other: TA97, TA102

Remarks:

Fujita et al. (1992)

Species / strain / cell type:

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

Remarks:

Haworth et al. (1983)

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA1538 and TA98

Remarks:

Heck et al. (1989)

Species / strain / cell type:

S. typhimurium, other: TA98 and TA100

Remarks:

Kasamaki et al. (1982)

Species / strain / cell type:

S. typhimurium, other: TA100, TA2637, TA98

Remarks:

Nohmi et al. (1985)

Species / strain / cell type:

S. typhimurium, other: TA98, TA100

Remarks:

Sasaki and Endo (1978)

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 was prepared from Aroclor 1254-induced male Fischer F344 rats and male B6C3F1 mice

Test concentrations with justification for top dose:

Dillon et al. (1998): 33 – 3333 microg/plate, top dose was limited by toxicity as determined by thinning of background lawn or a reduction in the number of colonies per plate (or both).
Florin et al. (1980) – A spot test at 3 micromol/plate was performed initially. If toxicity was observed, the definitive study was done at lower concentrations. Where the spot test did not demonstrate toxicity, the definitive test was run at 0.03, 0.3, 3.0 and 33.0 micromol/plate.
Fujita et al. (1992) – 0, 0.01, 0.05, 0.1, 0.5 and 1.0 mg/plate. No justification for dose range is given, and toxicity is not mentioned.
Haworth et al. (1983) – 0, 10.0, 33.0, 100.0, 333.0 and 1000.0 microg/plate, top dose limited by toxicity as determined in a preliminary toxicity screen.
Heck et al. (1989) – 37,500 nl/plate (corresponds to 3,900 microg/plate), toxicity was not pre-screened, but toxicity during main study was taken into consideration.
Kasamaki et al. (1982) – 0.05 to 5000.0 microg/plate, rationale for top dose is not specified.
Nohmi et al. (1985) – 0, 0.1, 0.2, 0.5, 1.0 and 2.0 mg/plate, top dose was limited by toxicity.
Sasaki and Endo (1978) – Information is in abstract form only, no details on concentrations available.

Vehicle / solvent:

Dillon et al. (1998) – vehicle is not mentioned.
Florin et al. (1980) – Ethanol
Fujita et al. (1992) – DMSO
Haworth et al. (1983) – Vehicle controls were run, but no specific mention of ID of vehicle for benzaldehyde.
Heck et al. (1989) – vehicle is not mentioned.
Kasamaki et al. (1982) – DMSO
Nohmi et al. (1985) – DMSO
Sasaki and Endo (1978) – vehicle is not mentioned.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

methylmethanesulfonate

mitomycin C

other: 2-Aminoanthracene used as + control for all strains w/ S9; without S9, formaldehyde or crotonaldehyde was used as + control for TA104.

Remarks:

Dillon et al. (1998)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Remarks:

Florin et al. (1980)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

mitomycin C

other: 2-aminoanthracene

Remarks:

Fujita et al. (1992)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-aminoanthracene and 4-nitro-o-phenylenediamine

Remarks:

Haworth et al. (1983)

Negative solvent / vehicle controls:

yes

Positive controls:

not specified

Remarks:

Heck et al. (1989)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

benzo(a)pyrene

other: aflatoxin B1

Remarks:

Kasamaki et al. (1982)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-aminoanthracene; 2-(2-furyl)-3-(5-nitrol-2-furyl)-acrylamide

Remarks:

Nohmi et al. (1985)

Negative solvent / vehicle controls:

not specified

Positive controls:

not specified

Remarks:

Sasaki and Endo (1978)

Details on test system and experimental conditions:

METHOD OF APPLICATION (Cell density at seeding, if applicable):
Dillon et al. (1998) – Preincubation, cell density not specified.
Florin et al. (1980) – per Ames et al. (1975)
Fujita et al. (1992) – per Maron and Ames (1983)
Haworth et al. (1983) – Preincubation, cell density not specified.
Heck et al. (1989) – per Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – per Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – per Ames et al. (1975)
Sasaki and Endo (1978) – Abstract only, references "procedure recommended by Ames et al."

DURATION
- Preincubation period:
Dillon et al. (1998) – per Maron and Ames (1983)
Florin et al. (1980) – per Ames et al. (1975)
Fujita et al. (1992) – per Maron and Ames (1983)
Haworth et al. (1983) – per Yahagi et al. (1975)
Heck et al. (1989) – per Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – per Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – per Ames et al. (1975)
Sasaki and Endo (1978) – Abstract only, references "procedure recommended by Ames et al."

- Exposure duration:
Dillon et al. (1998) – per Maron and Ames (1983)
Florin et al. (1980) – per Ames et al. (1975)
Fujita et al. (1992) – per Maron and Ames (1983)
Haworth et al. (1983) – 20 minutes with test material
Heck et al. (1989) – per Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – per Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – per Ames et al. (1975)
Sasaki and Endo (1978) – Abstract only, references "procedure recommended by Ames et al."

- Expression time (cells in growth medium):
Dillon et al. (1998) – per Maron and Ames (1983)
Florin et al. (1980) – per Ames et al. (1975)
Fujita et al. (1992) – per Maron and Ames (1983)
Haworth et al. (1983) – colonies counted 48 hours post pre-incubation.
Heck et al. (1989) – per Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – per Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – per Ames et al. (1975)
Sasaki and Endo (1978) – Abstract only, references "procedure recommended by Ames et al."

- Selection time (if incubation with a selection agent):
Dillon et al. (1998) – per Maron and Ames (1983)
Florin et al. (1980) – per Ames et al. (1975)
Fujita et al. (1992) – per Maron and Ames (1983)
Haworth et al. (1983) – per Yahagi et al. (1975)
Heck et al. (1989) – per Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – per Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – per Ames et al. (1975)
Sasaki and Endo (1978) – Abstract only, references "procedure recommended by Ames et al."

SELECTION AGENT (mutation assays):
Dillon et al. (1998) – per Maron and Ames (1983)
Florin et al. (1980) – per Ames et al. (1975)
Fujita et al. (1992) – per Maron and Ames (1983)
Haworth et al. (1983) – per Yahagi et al. (1975)
Heck et al. (1989) – per Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – per Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – per Ames et al. (1975)
Sasaki and Endo (1978) – Abstract only, references "procedure recommended by Ames et al."

NUMBER OF REPLICATIONS:
Dillon et al. (1998) – Three plates per dose
Florin et al. (1980) – not specified, assumed to follow Ames et al. (1975)
Fujita et al. (1992) – mean of three plates
Haworth et al. (1983) – mean of three plates
Heck et al. (1989) – not specified, assumed to follow Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – not specified, assumed to follow Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – not specified, assumed to follow Ames et al. (1975)
Sasaki and Endo (1978) – not specified

Evaluation criteria:

Dillon et al. (1998) – From section titled "Data Analyses": "The significance of mean revertant counts at individual dose levels was assessed using Dunnett's (-test and dose-response effects were analysed by two methods, Wahrendorf ranking and linear regression (Mahon et al, 1989). The latter was used to obtain slopes of the responses and both methods were only applied in the absence of a decline in revertant count at high dose levels. These analyses were used for guidance, but did not dictate the summary responses. Responses were judged mutagenic (+) when there was a reproducible, dose-related response and weakly mutagenic (+w) when reproducible, low level increases were obtained or when both positive and equivocal responses were seen in repeat trials. Responses were judged equivocal (?) when the increases in revertants were not reproducible or were seen only at a single dose.

Florin et al. (1980) – not detailed, assumed to follow Ames et al. (1975)

Fujita et al. (1992) – Statistical significance determined by Kruskal Wallis analysis.

Haworth et al. (1983) – from the section titled "Data Evaluation": "The data were evaluated in an ad hoc manner by each testing laboratory and by NTP personnel. Prior to statistical analysis no formal rules were used; however, a positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not."

Heck et al. (1989) – not detailed, assumed to follow Ames et al. (1973) and McCann et al. (1975)

Kasamaki et al. (1982) – from the section titled "Materials and Methods": "For mutagenic potency, a positive result was defined as a reproducible, dose-related increase in the number of revertant colonies per plate, and a greater than 2-fold increase in spontaneous mutation rate was obtained, according to the protocol of Ames (McCann et al., 1975)."

Nohmi et al. (1985) – at least 2-fold, dose dependent increase in mutant frequency.

Sasaki and Endo (1978) – no details.

Statistics:

See "Evaluation Criteria" section.

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Dillon et al. (1998)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

Dillon et al. (1998)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 104

Remarks:

Dillon et al. (1998)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Florin et al. (1980)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Florin et al. (1980)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Florin et al. (1980)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Florin et al. (1980)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

Remarks:

Fujita et al. (1992)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

Fujita et al. (1992)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Haworth et al. (1983)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Haworth et al. (1983)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Haworth et al. (1983)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Haworth et al. (1983)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Heck et al. (1989)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Heck et al. (1989)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Heck et al. (1989)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Heck et al. (1989)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Kasamaki et al. (1982)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Kasamaki et al. (1982)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA2637

Remarks:

Nohmi et al. (1985)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Nohmi et al. (1985)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Nohmi et al. (1985)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

Remarks:

Sasaki and Endo (1978)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Sasaki and Endo (1978)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Sasaki and Endo (1978)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Please see attached PDF tabular summary of all tests.

Conclusions:

While there is not a single study on in vitro bacterial mutagenicity which fully meets the criteria of the current OECD TG 471 (1997 version), there are several studies in the published literature performed in a manner similar to the guideline. These studies all either follow the original Ames design, or some minor variation. Most notably, there is not a single study which evaluates all recommended strains in the current OECD TG471, but the combined studies do cover the 5 combinations:

1. S. typhimurium TA1535, and
2. S. typhimurium TA1537 or TA97 or TA97a, and
3. S. typhimurium TA98, and
4. S. typhimurium TA100, and
5. E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102.

In addition, while only one study reached the recomended maximum exposure concentration of 5 mg/plate, one other was relatively close (3.3 mg), and the remaining studies' top doses were limited by toxicity.

The most notable thing is that not one of the 8 studies summarized in this endpoint study record gave a positive result for benzaldehyde. Taken together, these 8 studies can be considered to comprise a Weight of Evidence approach to demonstrating that benzaldehyde is not considered mutagenic when evaluated in an in vitro bacterial mutagenicity assay.

Executive summary:

Despite there not being one study that meets all of the criteria of the current OECD TG 471, the results of 8 studies performed in a manner similar to the guideline support that benzaldehyde is not mutagenic in an in vitro bacterial mutagenicity assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The test substance was not mutagenic in Salmonella gene mutation assays (Florin et al., 1980; Kasamaki et al., 1982; Haworth et al., 1983).

It exhibited genotoxic activity in the mouse lymphoma assay (McGregor et al., 1989) and in assays for sister chromatid exchanges in both Chinese hamster ovary (CHO) cells (Galloway etal., 1986). Induction of chromosomal aberrations by the test substance was also reported in Chinese hamster lung cells at a dose stated to be 50 nM (5.3 ng/ml) (Kasamaki et al., 1982); however, the National Toxicology Program (NTP), using concentrations of the test substance which were approximately 10,000 times higher, found no increase in aberrations in CHO cells (Galloway et al., 1986). This basic pattern of no mutagenic activity in bacterial systems but possible weak clastogenic effects in some mammalian cell assays is also reflected in test results from metabolites of the test substance, i.e., benzoic acid.

In in-vivo sex-linked recessive lethal mutation assays with Drosophila melanogaster the test substance was tested negative after oral and ip administration.

No adequate in vivo data are available that confirm the weakly positive results reported in in vitro tests.

Justification for selection of genetic toxicity endpoint
The study in mammalian cells was chosen over that in bacterial cells, as the higher level study.

Justification for classification or non-classification

The available data do not lead to classification for genotoxicity according to DSD and CLP.