Valeraldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The existing in vitro mutagenicity data for n-valeraldehyde provide partially contradictionary results. Negative results were obtained in the bacterial reverse mutation (Ames) assay, which is lacking one tester strain. Negative results were also obtained in an in vitro SCE in human lymphocytes and an in vitro UDS test in human hepatocytes. But, positive results in vitro were obtained in an UDS test in rat hepatocytes, an alkaline elution test in CHO cells and a HPRT-test in V79 cells.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication which meets basic scientific principles (limited reporting)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

yes

Remarks:

: limited reporting

Principles of method if other than guideline:

No guideline stated but the test method used is similar to OECD test guideline 482. Five n-alkanals were tested.

GLP compliance:

no

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

hepatocytes: primary cultures from livers of two human subjects

Details on mammalian cell type (if applicable):

- primary human hepatocyte cultures were isolated from apparently healthy fragments of human livers obtained from one male and one femal human subject. Liver fragments were discarded during the course of prescribed surgery. As checked by both macroscopic and histological examinations, these fragments were devoid of appreciable alterations.
- primary hepatocytes were isolated by collagenase perfusion. The portion of viable cells was 65% and 83% respectively.
- Type and identity of media: Williams' medium E

Test concentrations with justification for top dose:

0, 3, 10, and 30 mM (0, 0.26, 0.86, and 2.58 mg/mL); in addition 100 mM (8.61 mg/mL) for the determination of cytotoxicity

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

Migrated to IUCLID6: concentration: 5 mM (0.37 mg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 20 h; cells were simultaneously exposed to test compound and tritiated thymidine (10 µCi/mL [methyl-3H]thymidine))
- Expression time (cells in growth medium): 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

STAIN (for cytogenetic assays): hematoxylin and eosin (after development of autoradiographs)

NUMBER OF REPLICATIONS: two cultures from distinct rats each

NUMBER OF CELLS EVALUATED: total of 200; 100 (2 slides) per culture

DETERMINATION OF CYTOTOXICITY
- Method: determination of viability (percent survival) by counting cells (1000/dish) after staining exposed cell cultures with 0.4% trypan blue

Evaluation criteria:

A compound is considered positive if a dose-dependent increase is observed in net nuclear grains for at least two points and both increased points are above the justified laboratory-specific threshold, and/or statistical significance is demonstrated for both points (Internation Workshop on Standardization of Genotoxicity Test Procedures, Melbourne, 1992)
An absolute threshold for a positive response (i.e. 5 net nuclear grains) was not used.

Statistics:

Statistical significance compared to controls was assessed by Student's t-test (two-tailed)

Species / strain:

hepatocytes: primary cultures from livers of two human subjects

Metabolic activation:

not specified

Genotoxicity:

negative

Remarks:

see table below

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see table below

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results of UDS test with primary human hepatocytes

 

Treatment	Concentration [mg/mL]	% Cell survival		NUC* (mean ± SD)	CYT* (mean ± SD)	NNG* (mean ± SD)	% Repair**
		Subject 1	Subject 2
Controls		92	97	8.88 ± 3.36	8.33 ± 3.12	0.55 ± 1.69	1
Valeraldehyde	0.26	91	95	10.01 ± 3.33	9.16 ± 2.40	0.85 ± 2.07	6
	0.86	90	95	11.40 ± 4.21	10.54 ± 3.84	0.86 ±2.63	5
	2.58	87	91	9.09 ± 5.12	8.00 ± 4.25	1.09 ± 3.02	8
DMNA****	0.37	80	93	24.64 ± 5.38	6.51 ± 3.94	18.13 ± 4.95***	90

 

* NUC: nuclear grain count; CYT: cytoplasmic grain count; NNG:net nuclear grains

** Percentage repair is the percentage of cells with net nuclear labeling ≥ 5 grains

*** p < 0.001

**** DMNA: N,N-Dimethylnitrosamine

No increase of net nuclear grains was observed in valeraldehyde treated cell cultures. The number of cells in repair was always below 10%.  

Conclusions:

In a UDS test similar to OECD test guideline 482, no increase in DNA repair could be observed after treatment of primary human hepatocytes with valeraldehyde. Valeraldehyde was demonstrated to be negative and not to induce a mutagenic response in primary human hepatocytes.

Executive summary:

In an unscheduled DNA synthesis assay, primary cultures of human hepatocyte were exposed to valeraldehyde (purity 98%) at concentrations of 0, 0.26, 0.86, and 2.58 mg/ml for 20 h in the presence of tritium labeled thymidine. Human hepatocytes were obtained from discarded liver fragments during prescribed surgery of two human subjects.

 

Valeraldehyde was tested up to concentrations indicating the beginning of cytotoxicity. Treated cells did not show any statistically significant increases in DNA synthesis. The positive controls induced the appropriate response. There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures (nuclear silver grain counts) was induced. Primary cultures of human hepatocytes were demonstrated to be negative in this unscheduled DNA synthesis assay (Martelli 1994).

 

This study is classified as acceptable. It satisfies the requirement of Test Guideline OECD 482 for DNA damage and repair (deviation: limited reporting).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, adequately-documented publication which meets basic scientific principles (deviations from guideline: only tests without metabolic activation, only 3 concentrations tested, positive control substance differing from TG, limited reporting)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

only tests without metabolic activation, only 3 concentrations tested, positive control substance differing from TG, limited reporting

Principles of method if other than guideline:

Pre-guideline study, but the method used is similar to OECD test guideline 476 (In Vitro Mammalian Cell Gene Mutation Test). The mutagenic effect was examined for 2 target genes (HPRT locus - resistance to 6-thioguanine (TG) and Na/K-ATPase locus - resistance to ouabain (OUA)). Five n-alkanals were tested.

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT and Na/K-ATPase locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The cells for this study were obtained from NIH, Coriell Institute for Medical Research (Camden, NJ, USA)

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 3, 10, 30 mM (0.26, 0.86, 2.58 mg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N-nitrosourea (MNU; 0.3 and 1.0 mM)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation in serum free medium

DURATION
- Preincubation period: 60 min
- Exposure duration: 60 min
- Expression time (cells in growth medium): 6 days
- Selection time (if incubation with a selection agent): 8 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 10 petri plates

NUMBER OF CELLS EVALUATED: 3 x 10E05 per plate

DETERMINATION OF CYTOTOXICITY
- Method: other: relative survival

Evaluation criteria:

The test result is considered as positive (test substance mutagenic) if the mutant frequency induced by the test substance is threefold higher than the spontaneous mutant frequency (mutant frequency in negative controls).

Statistics:

Significance levels were determined according to Kastenbaum and Bowman (1970, Mutat Res 9, 527-549)

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

highest dose relative survival 56%

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Mutation Frequency and Relative Survival

 

Substance	Concentration [mM (mg/mL)]	HPGRT locus		Na/K-ATPase locus
		Mutation Frequency	Relative Survival	Mutation Frequency	Relative Survival
Valeraldehyde	0	13.3 ± 4.7	0.93 ± 0.01	1.8 ± 1.5	0.78 ± 0.07
	3 (0.26)	18.6 ± 2.7	0.87 ± 0.06	2.6 ± 2.5	0.89 ± 0.09
	10 (0.86)	36.7 ± 10.0**	0.73 ± 0.22	4.3 ± 3.1*	0.66 ± 0.26
	30 (2.58)	66.6 ± 18.5**	0.56 ± 0.26	6.5 ± 2.3**	0.44 ±0.13
MNU (pos. control)	0	15.1 ±0.3	0.89 ± 0.04
	0.3 (0.031)	38.1 ± 5.3**	0.84 ± 0.04
	1.0 (0.1)	106.8 ± 33.3**	0.64 ± 0.12
EMS (pos. control)	0			1.4 ± 0.8	0.81 ± 0.08
	8 (0.99)			18.1 ± 4.6**	0.69 ± 0.05
	16 (1.99)			28.4 ± 7.0**	0.59 ± 0.14

 

* P < 0.05 ** P < 0.01

 

Conclusions:

Interpretation of results: positive without metabolic activation

In a mammalian in vitro gene mutation test (HPRT assay), valeraldehyde induced a dose dependent, more than 3-fold, statistically significant increase in mutant colonies compared to negative controls and was demonstrated to be mutagenic in the test system under the test conditions used.

Executive summary:

In a mammalian cell gene mutation assay (forward mutation at the HPRT locus and the Na/K-ATPase locus), V79 cells (Chinese hamster lung fibroblasts) cultured in vitro were exposed to valeraldehyde (purity 98%) in the absence of mammalian metabolic activation at concentrations of 0, 0.26, 0.86, and 2.58 mg/mL.

 

Valeraldehyde was tested up to cytotoxic concentrations (highest dose 56% survival). At the highest dose, increase in mutation frequency was about fivefold of controls (66.6 versus 13.3) The positive controls did induce the appropriate response. There was a concentration related positive response of induced mutant colonies over background. In this test, valeraldehyde was demonstrated to show a positive mutagenic response under the test conditions used (Brambilla 1989).

 

This study is classified as acceptable. Though pre-guideline, this study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity data (mammalian forward gene mutation) with some restrictions (only tests without metabolic activation, only 3 concentrations tested, positive control substance differing from TG, limited reporting).

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication which meets basic scientific principles (limited reporting)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

yes

Remarks:

limited reporting

Principles of method if other than guideline:

No guideline stated but the test method used is similar to OECD test guideline 482. Five n-alkanals were tested.

GLP compliance:

no

Type of assay:

other: in vitro UDS assay

Species / strain / cell type:

hepatocytes: primary cultures from livers of male Sprague-Dawley rats

Details on mammalian cell type (if applicable):

- primary hepatocytes were isolated from rat liver by collagenase perfusion as described by Williams (1977, Cancer Res 37, 1845-1851). Cells were at least 80% viable.
- Type and identity of media: Williams' medium E

Test concentrations with justification for top dose:

0, 3, 10, and 30 mM (0, 0.26, 0.86, and 2.58 mg/mL); in addition 100 mM (8.61 mg/mL) for the determination of cytotoxicity

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

concentration: 5 mM (0.37 mg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 20 h; cells were simultaneously exposed to test compound and tritiated thymidine (10 µCi/mL [methyl-3H]thymidine))
- Expression time (cells in growth medium): 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

STAIN (for cytogenetic assays): hematoxylin and eosin (after development of autoradiographs)

NUMBER OF REPLICATIONS: two cultures from distinct rats each

NUMBER OF CELLS EVALUATED: total of 200; 100 (2 slides) per culture

DETERMINATION OF CYTOTOXICITY
- Method: determination of viability (percent survival) by counting cells (1000/dish) after staining exposed cell cultures with 0.4% trypan blue

Evaluation criteria:

A compound is considered positive if a dose-dependent increase is observed in net nuclear grains for at least two points and both increased points are above the justified laboratory-specific threshold, and/or statistical significance is demonstrated for both points (Internation Workshop on Standardization of Genotoxicity Test Procedures, Melbourne, 1992)
An absolute threshold for a positive response (i.e. 5 net nuclear grains) was not used.

Statistics:

Statistical significance compared to controls was assessed by Student's t-test (two-tailed)

Key result

Species / strain:

hepatocytes: primary culture from livers of male Sprague-Dawley rats

Metabolic activation:

not specified

Genotoxicity:

positive

Remarks:

see table below

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see table below

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: results for controls are within the range of historical control data

Results of UDS test with primary rat hepatocytes

 

Treatment	Concentration [mg/mL]	% Survival*	NUC** (mean ± SD)	CYT** (mean ± SD)	NNG** (mean ± SD)	% Repair***
Controls		96.3 ± 2.18	10.27 ± 4.52	9.64 ± 5.03	0.63 ± 4.76	5
Valeraldehyde	0.26	92.0 ± 3.60	11.00 ± 5.24	8.65 ± 3.05	2.35 ± 4.24****	26
	0.86	90.7 ± 5.03	14.44 ± 4.16	12.14 ± 5.10	2.30 ± 3.78****	23
	2.58	75.0 ± 22.72	15.13 ± 4.01	10.88 ± 4.25	2.65 ± 3.88****	22
	8.61	36.3 ± 31.94	-	-	-	-
DMNA*****	0.37	93.0 ± 5.14	40.66 ± 21.50	7.25 ± 5.1	33.41 ± 23.11****	91

 

* Data are means ± SD of three independent assays

** NUC: nuclear grain count; CYT: cytoplasmic grain count; NNG: net nuclear grains

*** percentage repair is the percentage of cells with net nuclear labeling ≥ 5 grains

**** p < 0.001

***** DMNA: N,N-Dimethylnitrosamine

Since valeraldehyde caused a marginal and not significant increase of cytoplasmic labeling, the increase in the number of net nuclear grains can definitely attributed to DNA repair synthesis. The occurrence of DNA repair is supported by the parallel increase in the number of cells in repair.

Conclusions:

Interpretation of results:
positive response is only weak positive

In a UDS test similar to OECD test guideline 482, a statistically significant increase in DNA repair (about 4-fold of controls) was observed after treatment of primary rat hepatocytes with valeraldehyde. Though the response was weak and did not reach the value of 5 net nuclear grains, the effect is considered as positive.

Executive summary:

In an unscheduled DNA synthesis assay, primary rat hepatocyte cultures were exposed to valeraldehyde (purity 98%) at concentrations of 0, 0.26, 0.86, and 2.58 mg/ml for 20 h in the presence of tritium labeled thymidine.

 

Valeraldehyde was tested up to cytotoxic concentrations. Treated cells showed statistically significant increases in DNA synthesis (controls 0.63 NNG, treated cells between 2.30 and 2.65 NNG; p < 0.001). The positive controls induced the appropriate response (33.41 NNG). There was evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures (nuclear silver grain counts) was induced but only to a very moderate extent (Martelli 1994).

 

This study is classified as acceptable. It satisfies the requirement of Test Guideline OECD 482 for DNA damage and repair (deviation: limited reporting).

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

only 4 strains of S. thyphimurium tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 strains of S. thyphimurium tested

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

S. typhimurium, other: TA97, TA 98, TA 100, TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from livers of induced maled Sprague Dawley rats and induced male Syrian hamsters (10% and 30% each)

Test concentrations with justification for top dose:

0, 10, 33, 100, 333, 1000, 2000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: 9-aminoacridine (TA97), sodium azide (TA 100, TA 1535), 2-nitrofluorene (TA 98); with metabolic activation: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days at 37°C

DETERMINATION OF CYTOTOXICITY
- Method: no data

Evaluation criteria:

Number of mutant colonies of positive controls must be significantly increased over the spontaneous control numbers for the test to be considered valid.
Numbers of mutant colonies of test plates have to be significantly increased over the mutant colony numbers of negative control plates.

Statistics:

Mean and standard error of the mean of replicate plates were calculated

Species / strain:

S. typhimurium, other: TA97, TA 98, TA 100, TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting from a concentration of ca. 1000 µg/plate depending on strain and experimental conditions (metabolic system)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Tables presenting the revertant counts for all tests including information on cytotoxicity are appended under attached background material.

Conclusions:

Interpretation of results:
negative with and without metabolic activation

n-Pentanal (valeraldehyde) was not mutagenic in Salmonella typhimurium strains TA 97, TA 98, TA100 and TA1535 with and without metabolic activation.
According to OECD 471, valeraldehyde is not mutagenic to Salmonella typhimurium.

Executive summary:

In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA 97, TA 98, TA 100, and TA 1535) were exposed to n-pentanal (valeraldehyde) at concentrations of 0, 10, 33, 100, 333, 1000, and 2000 µg/plate in the presence and absence of mammalian metabolic activation (S9 mix from induced rabbit and rat liver). Preincubation was 20 min before plating.

Valeraldehyde was tested up to the limit concentration of 2000 µg/plate. With the two highest concentrations, fractional cytotoxicity was observed depending on strain and metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. Valeraldehyde did not increase the number of revertants in any of the test strains. There was no evidence of induced mutant colonies over background (NTP, 1988).

 

This study is classified as acceptable. It was performed according to OECD test guideline 471 with minor restrictions (only 4 strains of S. thyphimurium tested).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No in vivo genotoxicity studies have been identified with n-valeraldehyde. Negative results were obtained in an in vivo micronucleus assay with the structural similar iso-valeraldehyde, which is considered to be representative for valeraldehyde.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-10-09 - 2001-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Remarks:

Experimental Toxicology and Ecology, BASF AG

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Deutschland
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: mean ca. 28 g
- Assigned to test groups randomly: yes
- Housing: housed individually in Makrolon cages, type MI
- Diet (e.g. ad libitum): ad libitum, Kliba Haltungsdiät, Provimi Kliba SA, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): ad libitum, drinking water from bottles
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
All test substance formulations were prepared immediately before administration

Duration of treatment / exposure:

single injection

Frequency of treatment:

one single application

Post exposure period:

24, 48 h

Remarks:

Doses / Concentrations:
0, 25, 50, 100 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPP), 20 mg/kg body weight for clastogenic effects
Vincristirie Sulphate (VCR), 0,15 mg/kg body weight for aneugenic effects
- Justification for choice of positive control(s): The stability of CPP and VCR is well-defined under the selected conditions, since both positive control articles are well-defined clastogens and aneugens respectively
- Route of administration: intraperitoneally

Tissues and cell types examined:

polychromatic and normochromatic erythrocytes of bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity, deaths were observed down to a dose of 150 mg/kg body weight. 100 mg/kg body weight were survived by all animals but led to evident signs of toxicity such as piloerection, squatting posture, and the general state of the animals was poor, however, showing no distinct symptomatic differences between the male and female animals.
Thus, only male animals were used for the cytogenetic investigations.
Therefore, a dose of 100 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 50 mg/kg and 25 mg/kg body weight were administered as further doses.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were treated once and samples of bone marrow were taken 24 hours and 48 hours after the treatment
- The low dose group was given 25 mg test substance/kg body weight or 4 ml/kg body weight of a solution with a concentration of 0.625 g/100 ml.
- The intermediate dose group was given 50 mg test substance/kg body weight or 4 ml/kg body weight of a solution with a concentration of 1.25 g/100 ml.
- The top dos~: groups were given 100 mg test substance/kg body weight or 4 ml/kg body weight of a solution with a concentration of 2.5 g/100 ml

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by SCHMID (1976):
- The two femora were prepared by dissection and removing all soft tissues.
After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 pl fresh FCS. 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

METHOD OF ANALYSIS:
In general, 2000 polychromatic erythrocytes (PCE) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur are also scored.

Evaluation criteria:

The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-relatod and significant increase in the number of micronucleated polychromatic erythrocytes at any of the intervals.
- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test system if:
- There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time.
- The frequencies of cells containing micronuclei were within the historical control range.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft) .
The number of micronuclei in polychromatic erythrocytes was analyzed. A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p <= 0.05, ** for p >= 0.01) were printed with the group means in the tables.
This test was performed one-sided.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

squatting, poor general state, piloerection

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 150, 100 mg/kg bw
- Clinical signs of toxicity in test animals: piloerection, squatting posture, and the general state of the animals was poor

The administration of the test substance led to evident signs of toxicity:
- 25 mg/kg bw:
 1, 2 and 4 h: squatting posture; 1 d: no symptoms
- 50 mg/kg bw:
 1 h: squatting posture; 2 and 4 h: squatting posture and  poor general state; 1 d: no symptoms
- 100 mg/kg bw:
 1 h: squatting posture and poor general state, 2 and 4 h:
 squatting posture, poor general state and piloerection;
 1 d: poor general state and piloerection

 

Results:

Test group	Interval: 24 h				Interval: 48 h
	Polychromatic erythrocytes	Normocytes / total amount polychromatic erythrocytes	Cells with micronuclei (%)		Polychromatic erythrocytes	Normocytes / total amount polychromatic erythrocytes	Cells with micronuclei (%)
Dose (mg/kg bw)			polychromatic	normochromatic			polychromatic	normochromatic
vehicle control	10000	3396	1.3	0.3	100000	2974	1.7	1.0
25	10000	3997	1.3	1.5
50	10000	3764	2.2	2.9
100	10000	3769	1.9	1.6	10000	4193	0.9	1.0
20, cyclophosphamide	10000	3092	21.5**	3.6
0.15, vincristine	10000	4859	49.7**	2.3

* p<=0.05; ** p<= 0.01

 

There was no increase in the number of polychromatic erythrocytes containing either small or large micronuclei.
The rate of microneuclei was nearly the range of the concurrent negative control in all dose groups and within the range of the historical control data.
No inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected.

The result for the negative control was within the historical control range.

Both of the positive control chemicals, i.e. cyclophosphamide for clastogenic effects and vincristine for induction of spindle poison effects, induced the expected increases in the rate of polychromatic erythrocytes containing small or large micronuclei.

Conclusions:

Interpretation of results: negative
The test substance had no chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Executive summary:

In this in vivo micronucleus test (according to OECD Guideline 474, GLP), 5 male mice (strain: NMRI) were dosed intraperitoneally with the test item isovaleraldehyde. Test doses were 0, 25, 50, 100 mg/kg bw. The top dose was selected based on a pretest were mortality was observed down to a dose of 150 mg/kg bw and 100 mg/kg bw was survived by all animals but led to signs of toxicity. The animals were treated once and samples of bone marrow were taken 24 hours and 48 hours after the treatment. In general, 2000 polychromatic erythrocytes (PCE) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur were also scored.

There was no increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of microneuclei was nearly the range of the concurrent negative control in all dose groups and within the range of the historical control data. No inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected.

The result for the negative control was within the historical control range. According to this study, the test item is considered to be not chromosome-damaging (clastogenic) effect, and does not lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity).

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For details on endpoint specific justification please see read-across justification report attached to this endpoint study record.

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

squatting, poor general state, piloerection

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

The administration of the test substance led to evident signs of toxicity:
- 25 mg/kg bw:
 1, 2 and 4 h: squatting posture; 1 d: no symptoms
- 50 mg/kg bw:
 1 h: squatting posture; 2 and 4 h: squatting posture and  poor general state; 1 d: no symptoms
- 100 mg/kg bw:
 1 h: squatting posture and poor general state, 2 and 4 h:
 squatting posture, poor general state and piloerection;
 1 d: poor general state and piloerection

 

Results:

Test group	Interval: 24 h				Interval: 48 h
	Polychromatic erythrocytes	Normocytes / total amount polychromatic erythrocytes	Cells with micronuclei (%)		Polychromatic erythrocytes	Normocytes / total amount polychromatic erythrocytes	Cells with micronuclei (%)
Dose (mg/kg bw)			polychromatic	normochromatic			polychromatic	normochromatic
vehicle control	10000	3396	1.3	0.3	100000	2974	1.7	1.0
25	10000	3997	1.3	1.5
50	10000	3764	2.2	2.9
100	10000	3769	1.9	1.6	10000	4193	0.9	1.0
20, cyclophosphamide	10000	3092	21.5**	3.6
0.15, vincristine	10000	4859	49.7**	2.3

* p<=0.05; ** p<= 0.01

 

There was no increase in the number of polychromatic erythrocytes containing either small or large micronuclei.
The rate of microneuclei was nearly the range of the concurrent negative control in all dose groups and within the range of the historical control data.
No inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected.

The result for the negative control was within the historical control range.

Both of the positive control chemicals, i.e. cyclophosphamide for clastogenic effects and vincristine for induction of spindle poison effects, induced the expected increases in the rate of polychromatic erythrocytes containing small or large micronuclei.

Conclusions:

Interpretation of results: negative
The test substance had no chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Executive summary:

The study used as source investigated the effect of isovaleraldehyde on genotoxicity. The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

In the absence of information on a species specific mode of action the available information is regarded as relevant for humans.

Additional information

The genotoxic potential of valeraldehyde was tested in vitro with bacterial and mammalian cells (see table below). In vivo genotoxicity studies could not be identified. However, anin vivo test results is available for the closely related isovaleraldehyde (structural isomer with a branched carbon chain) which can be used for read across. 

 

The following table summarises the available studies used in the genetic toxicity assessment of valeraldehyde:

 

 

Study	Substance	Reliability	GLP	Test type/ Protocol	Guideline	Results	Remarks
In vitro tests; gene mutation studies in bacteria
NTP 1988	Valeraldehyde	2	no data	Ames	similar OECD 471	negative	tested up to 2000 µg/plate with and without metab. activ.
In vitro tests, studies in mammalian cells
Brambilla 1989	Valeraldehyde	2	no	HPRT gene mutation	similar OECD 476	positive	V79 cells; without metabolic activation; 3 concentrations up to 2580 µg/mL; cytotoxicity observed
Martelli 1994	Valeraldehyde	2	no	Gen. Tox.  UDS	similar OECD 482	positive	primary hepatocytes; 3 concentrations, up to 2580 µg/mL cytotoxicity observed
Marinari 1984	Valeraldehyde	2	no	DNA damage (alkaline elution)	no TG	positive	single strand breaks, no cross linking
In vivo tests; somatic cell studies
BASF AG 2001	Read across; Isovaleraldehyde	1	yes	MNT	Similar to OECD 474	negative	Read across; single ip injection; 3 concentrations up to 100 mg/kg bw (overt toxicity)

   

 

The Ames test is negative.

 

Results of in vitro tests with mammalian cell systems are predominantly positiv.

In vivo genotoxicity studies for valeraldehyde are not available, but data from the closely related iso-valeraldehydes can be used for read across.

 

Isovaleraldehyde was tested by BASF in an in vivo mammalian erythrocyte micronucleus test. The result was negative (no increase in polychromatic erythrocytes containing neither small nor large micronuclei). The study satisfies the requirements of OECD TG 474.

   

Based on the above, it is concluded that valeraldehyde does not induce cytogenic effects. But the positive results in the mammalian cells require further clarification.

Justification for classification or non-classification

Based on the available information it is concluded that valeraldehyde does not meet classification criteria according to Regulation (EC) No 1272/2008 for germ cell mutagenicity.