Benzaldehyde

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Benzaldehyde

EC Number:

202-860-4

EC Name:

Benzaldehyde

Cas Number:

100-52-7

Molecular formula:

C7H6O

IUPAC Name:

benzaldehyde

Test material form:

liquid

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

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.