Propionaldehyde, reaction product with formaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro chromosome aberration

In vitro Mammalian Chromosome Aberration Test, OECD 473, EU Method B.10: positive/clastogenic

In vitro gene mutation in bacteria

Bacterial Reverse Mutation Assay, OECD 471, EU method B.13/14: positive/mutagenic in Salmonella typhimurium TA97a, TA100 and TA102 with and without an external metabolising system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 1 December 2009 to 10 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Lyophilized Post-Mitochondrial Supernatant (Moltox, Art. No. 11-01L.2, Lot.: 2361) from male Sprague-Dawley rat livers, induced with Aroclor-1254, in 0.154M KCL

Test concentrations with justification for top dose:

Experiment A (3 hours incubation, no metabolic activation system): 0.044, 0.13, 0.39, 1.18 mg/mL
Experiment A (3 hours incubation, with metabolic activation system): 0.044, 0.13, 0.39, 1.18 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium with L-glutamine (Gibco BRL Life Technologies, UK, article number 21875-034).
For each experiment a stock solution of the test substance was prepared in RPMI medium. To achieve the intended final concentrations of the test substance, the further test substance solutions (one for each tested concentration) were then obtained by diluting these stock solutions with RPMI medium.
All preparations were made freshly before adding them to the cell cultures.

- Justification for choice of solvent/vehicle: Common vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

see below for details Migrated to IUCLID6: and cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Cultures were kept at 37 °C and 5% CO2 for ca. 48 hours before further processing
- Exposure duration: 3 hours (one experiment without and one experiment with metabolic activation)
- Expression time (cells in growth medium): 15 hours (plus 2 hours with spindle inhibitor added)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): 0.1 mL of Colcemid (Gibco BRL Life Technologies, UK, article no 15210-057, 10 µg/mL in balanced salt solution) per culture.
STAIN (for cytogenetic assays): 10 minutes with Giemsa solution (10% v/v in a buffer of 0.067 M KH2PO4 and 0.067 M Na2HPO4 x 2 H2O in deionised water), rinsed in tap-water and then in deionised water.

NUMBER OF REPLICATIONS: 2 per culture

NUMBER OF CELLS EVALUATED:
Mitotic indices: 2000 lymphocytes per culture
Chromosome aberrations: 100 metaphases per culture (i.e. 200 per concentration, apart from cultures with obviously high numbers of metaphases with aberrations)

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic indices were determined by counting a total of 2000 lymphocytes per cell culture and by recording the number of lymphocytes in any stage of mitosis. This number was then expressed as percentage of mitotic lymphocytes.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Only well spread cells with 44 to 47 chromosomes, polyploid and endoreduplicated cells were acceptable for analysis. Structural aberrations were scored according to well defined (and reported) criteria.

Statistics:

The Chi2-Test (two-tailed, p=0.05) was used for the comparison between the negative control and the test substance cultures. If the results were positive, comparisons were made separately between the negative control and each concentration. If conditions for the Chi2-Test were not met, Fisher’s Exact Test was used. Chi2-Test or Fisher’s Exact Test were also used for the comparison between the negative and the positive controls.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- None

COMPARISON WITH HISTORICAL CONTROL DATA:
Yes, historical control data (on negative and positive controls) given in the report. The positive control substances caused in each experiment clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system, thus demonstrating that the test systems were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
see "any other information on results incl. tables"

Mitotic index

At the highest concentration of 1.18 mg/mL marked cytotoxicity was noted regardless whether a metabolic activation system was used or not.

Experiment without a metabolic activation system, 3 hours of incubation:

Test substance concentration (mg/mL)	0.044	0.13	0.39	1.18
Mitotic index (% of respective negative control)	114.5	115.3	96.5	54.0

Bold figures: These concentrations were analysed

 

Experiment with a metabolic activation system, 3 hours of incubation:

Test substance concentration (mg/mL)	0.044	0.13	0.39	1.18
Mitotic index (% of respective negative control)	103.0	73.4	82.0	23.1

Bold figures: These concentrations were analysed

Numerical aberrations No statistically significant differences in the number of metaphases with numerical aberrations were noted in any experiment at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.   Gaps In the experiment without a metabolic activation system the number of gaps was statistically significantly higher at all test substance concentrations compared to the corresponding negative control and there was no clear concentration-response relationship. In the experiment with a metabolic activation system no statistically significant increase in the number of gaps was noted at any concentration analysed compared to the concurrent negative controls. Structural aberrations In the experiment without a metabolic activation system the number of metaphases with structural aberrations was statistically significantly higher at all test substance concentrations compared to the corresponding negative control and there was a clear concentration-response relationship. The numbers were also clearly beyond the data of historical negative controls. In the experiment with a metabolic activation system the number of metaphases with structural aberrations was not statistically significantly increased at any concentration analysed compared to the concurrent negative controls. Nevertheless, at the highest concentration evaluated (0.39 mg/mL) the number of metaphases with structural aberrations was beyond the range of the historical negative controls and within the range of historical positive controls. The numbers of the other test substance concentrations (0.044 mg/mL and 0.13 mg/mL) were within the range of historical negative controls.

Conclusions:

The overall results of the study indicate clastogenic properties of the test substance at a treatment length of 3 hours without the use of a metabolic activation system. There is also relevant evidence that the test substance has mutagenic properties at a treatment length of 3 hours with the use of a metabolic activation system.

Executive summary:

The study was performed to determine possible mutagenic properties of test substance by means of an in vitro mammalian chromosome aberration test in human lymphocytes, according to Regulation (EC) No 440/2008 Method B.10. and to OECD Guideline 473.

A total of two experiments was performed and analysed: one of them without and one with the use of a metabolic activation system (liver microsomes from Aroclor 1254 induced rats, with a co-factor solution). A concentration range betweennominal 0.044 mg/mL and 1.18 mg/mL mg test substance per mL medium was tested. 1.18 mg/mL as the highest test substance concentration is 0.01M and was chosen in accordance with the EC directive and the OECD guideline.

Primary lymphocyte cultures were established from whole blood freshly obtained from one male donor. After 48 hours of incubation, the test substance was added. In all experiments, regardless whether a metabolic activation system was used or not, the test substance was washed out three hours later and the cultures were cultivated for another 17 hours. Colcemid was added two hours before the end of the cultivation period, and then cells were fixed and slides prepared.

The test substance was dissolved in RPMI medium and for each concentration of the test substance two cultures were established. One negative control (RPMI medium) and one positive control (methanesulfonic acid methyl ester, MMS, for cultures without metabolic activation system and cyclophosphamide, CP, for cultures with a metabolic activation system) were set up concurrently in each experiment.

The concentrations of the test substancein the experiments performed were 0.044, 0.13, 0.49 and 1.18 mg/mL.

In general, apart from cultures with obviously high numbers of metaphases with aberrations, 100 metaphases per culture (i.e. 200 per concentration) were analysed for structural and numerical chromosomal aberrations. The slides were coded before analysis. The mitotic indices were calculated from 2000 lymphocytes per culture for an assessment of cytotoxicity.

Results

Cytotoxicity

At the highest concentration of 1.18 mg/mL marked cytotoxicity was noted regardless whether a metabolic activation system was used or not.

Experiment without a metabolic activation system, 3 hours of incubation:

Test substance concentration (mg/mL)	0.044	0.13	0.39	1.18
Mitotic index (% of respective negative control)	114.5	115.3	96.5	54.0

Bold figures: These concentrations were analysed

 

Experiment with a metabolic activation system, 3 hours of incubation:

Test substance concentration (mg/mL)	0.044	0.13	0.39	1.18
Mitotic index (% of respective negative control)	103.0	73.4	82.0	23.1

Bold figures: These concentrations were analysed

Numerical aberrations

No statistically significant differences in the number of metaphases with numerical aberrations were noted in any experiment at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

Gaps

In the experiment without a metabolic activation system the number of gaps was statistically significantly higher at all test substance concentrations compared to the corresponding negative control and there was no clear concentration-response relationship.

In the experiment with a metabolic activation system no statistically significant increase in the number of gaps was noted at any concentration analysed compared to the concurrent negative controls.

Structural aberrations

In the experiment without a metabolic activation system the number of metaphases with structural aberrations was statistically significantly higher at all test substance concentrations compared to the corresponding negative control and there was a clear concentration-response relationship. The numbers were also clearly beyond the data of historical negative controls.

In the experiment with a metabolic activation system the number of metaphases with structural aberrations was not statistically significantly increased at any concentration analysed compared to the concurrent negative controls. Nevertheless, at the highest concentration evaluated (0.39 mg/mL) the number of metaphases with structural aberrations was beyond the range of the historical negative controls and within the range of historical positive controls. The numbers of the other test substance concentrations (0.044 mg/mL and 0.13 mg/mL) were within the range of historical negative controls.

As experiment A without metabolism gave positive results the performance of an experiment B with without a metabolic activation system with an extended treatment length of 20 hours, equivalent to about 1.5 normal cell cycle lengths was not required. Due to the clear positive result in the experiment without metabolism and due to the relevant evidence that the test substance did also induce structural chromosomal aberrations in the experiment with metabolism a confirmation of the experiment with metabolism was not considered to be necessary.

Positive controls

The positive control substances caused in each experiment clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system, thus demonstrating that the test systems were adequate and that the metabolic activation system functioned properly.

 

Conclusion

In each experiment the test substance caused marked cytotoxic effects (reduction to about 50% or less, compared to concurrent negative controls) at the highest concentration tested. The highest tested concentration was 0.01M and thus in accordance with the EC directive and the OECD guideline.

Under the conditions of this study the test substance induced structural chromosomal aberrations in cultured human lymphocytes after a treatment length of 3 hours in the absence of a metabolic activation system. The conclusion is based on a statistically significant increase of metaphases with structural aberrations at all test substance concentrations (1.18, 0.39 and 0.13 mg/mL) with a clear concentration-response relationship and on the fact that these figures were far beyond the range of historical negative controls. Gaps are not necessarily an indicator for clastogenic effects, but at all test substance concentrations the number of gaps was also statistically increased.

There was also relevant evidence that the test substance did induce structural chromosomal aberrations in cultured human lymphocytes in the experiment with metabolism, although there was no statistically significant increase compared to the controls. The conclusion is mainly based on an important increase of metaphases with structural aberrations at the highest test substance concentration (0.39 mg/mL) and on the fact that this figure was beyond the range of historical negative controls and within the range of historical positive controls.

The overall results of the study indicate clastogenic properties of the test substance at a treatment length of 3 hours without the use of a metabolic activation system. There is also relevant evidence that the test substance has mutagenic properties at a treatment length of 3 hours with the use of a metabolic activation system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 22 December 2009 to 18 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his- (s. typhimurium)

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

5000, 1667, 556, 185, 62 µg/plate

Vehicle / solvent:

deionised water

Details on test system and experimental conditions:

First experiment: Plate Incorporation Assay:
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution and
• 2 mL of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

Second experiment: Preincubation Assay:
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution.
The solutions were preincubated for 20 minutes at 37 °C using a shaker, afterwards combined with 2 mL of top agar and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

Evaluation criteria:

Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result were:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
•For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: 250 % of the amount of the spontaneous revertants.
•For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: 167 % of the amount of the spontaneous revertants.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRECIPITATION:
No precipitation of the test substance was seen in any of the concentration groups.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary test no toxicity was seen up to 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
The numbers of spontaneous revertants were comparable with the historic control data for the negative controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was seen up to 5000 µg/plate.

Conclusions:

According to these results obtained in this study, the test substance is mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA100 and TA102 with and without an external metabolising system.

Executive summary:

The test substance was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD Guideline 471 and the Council Regulation (EC) No 440/2008, Method B.13/14.

The test substance was dissolved in deionised water. The following concentrations were tested:

62,185, 556,1667 and 5000 µg per plate without external metabolisation, and

62,185, 556,1667and 5000 µg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system.

In the first experiment the test was performed according to the "direct plate incorporation method", in the second experiment according to the "preincubation method". As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included.

 

Results

Positive controls:

All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.

 

Test substance:

Toxicity:

No toxicity of the test substance to the bacteria was observed up to 5000 µg per plate.

 

Solubility:

No precipitation of the test substance was seen in any of the concentration groups.

Mutagenicity:

In the plates of the strains TA97a, TA100 and TA102 an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained in the higher concentrations.

In the strains TA98 and TA1535 no significant increase of the revertant counts was obtained.

Conclusion

 

According to these results obtained in this study, the test substance is mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA100 and TA102 with and without an external metabolising system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo cytogenicity / erythocyte micronucleus

Bone marrow micronucleus test in rats, OECD 474: negative

In vivo DNA damage and/or repair

Mammalian alkaline Comet Assay, OECD 489, negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 13 July 2020 to 16 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

All deviations that occurred during the study have been authorized/acknowledged by the Study Director, assessed for impact, and documented in the study records. All study plan deviations and those SOP deviations that could have impacted the quality or integrity of the study are listed below. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions. Animal Husbandry; Environmental Conditions -Temporary deviations from the maximum level of target humidity occurred. Evalution: This study plan deviation is considered not to have affected the integrity of the study because it did not noticeably affect the clinical condition of the animals or the outcome of the study. Main Study - In the positive control group in liver, due to a technical error animal 23 and 24 were combined. Furthermore less then 150 cells were analyzed in the positive control slides. Evaluation: As the positive control was clearly positive and within the historical control data range no impact on the study outcome was present. Comet Slide Analysis - In liver slides the examination of 150 cells could not always be performed due to a lower cell harvest. Evaluation: In the vehicle and test item (low, mid, high) groups 674, 657, 393 and 565 cells respectively were analyzed. As the test item clearly did not induce changes in tail intensity and the negative and positive control were within historical control data ranges this was not considered of impact on the study outcome.

Deviations:

yes

Remarks:

None of the deviations impact the study outcome.

GLP compliance:

yes

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

- Identification: Propionaldehyde, reaction product with formaldehyde (EC No 701-281-9 / CAS No -)
- Expiry date: 05 May 2022
- Physical Description: Colourless liquid
- Purity/Composition: UVCB
- Storage Conditions: At room temperature (do not store above 40°C)
- Test Facility test item number: 211302/A
- Purity/Composition correction factor: No correction factor
- Test item handling: No specific handling conditions required
- Chemical name (IUPAC, synonym or trade name): Propionaldehyde, reaction product with formaldehyde
- EC number: EC 701-281-9
- CAS number -
- Molecular formula: C5H10O3
- Molecular weight: 118.1326
- Stability at higher temperatures: Stable
- Specific gravity / density: 1.29 g/cm3

Species:

rat

Strain:

Wistar

Remarks:

Crl: Wistar (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Condition: Outbred, SPF-quality
- Age at study initiation: 6 weeks
- Weight at study initiation: 190 +/-10 g for males and 172-210 g for females
- Assigned to test groups randomly: yes, the animals were allocated at random to treatment groups.
-Animal identification: The rats were identified with a unique number on the tail written with marker pen
- Fasting period before study: no
- Housing: Up to 5 animals of the same sex and same dosing group together in the Polycarbonate cages (Makrolon MIV type; height 18 cm.) containing
sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany, Pellets ad libitum / Pellets / Ad libidum, except during designated procedures
- Water: Municipal tap water, Freely available ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Light cycle: 12 hours light and 12 hours dark (except during designated procedures)
- Air changes (per hr): 10 or more
The actual daily mean temperature during the study period was 19.4 to 20.5°C with an actual daily mean relative humidity of 53 to 74%.

Route of administration:

oral: gavage

Vehicle:

Milli-Q water (Millipore Corp., Bredford, MA, USA)
The specific gravity of Milli-Q water is 1.0 g/mL.

Details on exposure:

A limited quantity of food was supplied during the night before dosingm (approximately 7 g/rat).
The first day of dosing was designated as Day 1. The doses were given using stainless steel ball-tipped syringes. Animals received a single dose on two consecutive days. The dosing volume was 10 mL/kg body weight.
Test item concentrations were prepared on the day of administration.

Duration of treatment / exposure:

Animals received a single dose on two consecutive days.

Frequency of treatment:

Twice

Post exposure period:

two days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Test item

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Test item

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Test item

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

EMS

No. of animals per sex per dose:

Males: 3 (dose-range finding), 25 (main study - 5/group)
Females: 3 (dose-range finding)

Control animals:

yes

Positive control(s):

The positive control was ethyl methanesulfonate (EMS, Sigma Aldrich, Steinheim, Germany, batch BCBZ8402) at 200 mg/kg body weight dissolved in physiological saline. EMS was used within 2 hours after preparation and the route of administration was oral. The dosing volume was 10 mL/kg body weight.

Tissues and cell types examined:

liver, glandular stomach and duodenum cells, respectively.

Details of tissue and slide preparation:

Part of the liver, stomach and duodenum from the animals used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).

Evaluation criteria:

COMET SCORING:
To prevent bias, slides were randomly coded (per tissue) before examination of the comets. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample.
The following criteria for scoring of Comets were used:
- Only horizontal orientated Comets were scored, with the head on the left and the tail on
the right.
- Cells that showed overlap or were not sharp were not scored.
In addition, the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal in the repeat experiment. The occurrence of hedgehogs was scored in all treatment groups and the control. Since there was no effect of the test item Hedgehogs data was not reported and maintained in the raw data.

ACCEPTABILITY CRITERIA:
The in vivo comet is considered acceptable if it meets the following criteria:
- The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
- The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database. The positive control data was analysed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).
- Adequate numbers of cells and doses have been analysed
- The highest test dose is the MTD or 2000 mg/kg/day
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data .
A test item is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent
negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05)
increase in percentage Tail Intensity is detected compared with the concurrent negative
control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MORTALITY AND GENERAL CLINICAL OBSERVATIONS:
The animals of the groups treated with the test item and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality over the course of the in-life phase. Animal 5 showed situs inversus during necropsy. The mean body weights per group recorded immediately prior to dosing are presented in Table 1.
CLINICAL CHEMISTRY:
No test item-related changes in clinical chemistry parameters were noted in any of the test item treated groups. A slightly lower aspartate aminotransferase (ASAT) activity was observed in males at 500, 1000 and 2000 mg/kg. In absence of a dose-related response and at the magnitude of change, this finding was considered to be not toxicologically relevant. Furthermore, a slight increase in creatinine concentration was noted in males at 1000 mg/kg. In absence of a dose-related response, this finding was considered to be not test item-related.
In conclusion, no relevant test item-related clinical chemistry changes were observed in males up to 2000 mg/kg.
COMET SLIDES ANALYSIS:
No statistically significant increase in the mean Tail Intensity (%) was observed in liver (Table 2), glandular stomach (Table 3) and duodenum cells (Table 4) of test item treated male treated animals compared to the vehicle treated animals. No hedgehogs were observed in any of the slides.
The mean Tail Intensity in liver, glandular stomach and duodenum cells of vehicle-treated rats was 2.0 ± 0.85% (mean ± SD), 3.9 ± 1.3% (mean ± SD) and 4.5 ± 0.72% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 82 ± 2.0% (mean ± SD, 42-fold induction; p<0.001 Students t test;), 55 ± 4.0% (mean ± SD, 14-fold induction; p<0.001 Welch t test;) and 40 ± 6.1% (mean ± SD, 9.0-fold induction; p<0.001 Students t test;) in male animals in liver, glandular stomach and duodenum cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.
Adequate numbers of cells (150 cells per animal) and doses were analyzed in glandular stomach and duodenum and the highest test dose was the maximum dose required by the guidelines. In liver slides, the examination of 150 cells could not always be performed due to a lower cell harvest (see Study Plan Deviation). In the vehicle and test item (low, mid, high) groups 674, 657, 393 and 565 cells respectively were analyzed. As the test item clearly did not induce changes in tail intensity and the negative and positive control were within historical control data ranges this was not considered of impact on the study outcome. Hence, all criteria for an acceptable assay were met.

Table 1: Body weight immediately prior to dosing:  

Group code	Dose (mg/kg/bw)	Day 1 Body weight gram (Mean ± S.D.)			Day 2 Body weight gram (Mean ± S.D.)
1	0	193.8	±	10.9	189.4	±	9.2
2	500	188.0	±	4.5	183.6	±	3.9
3	1000	187.4	±	9.3	185.0	±	11.2
4	2000	189.0	±	15.7	185.8	±	13.4
5	200 (EMS)	190.2	±	12.5	184.4	±	11.4

Table 2: Overview Tail Intensity in liver Cells of Male Rats  

	Tail Intensity (%)	S.D.
Vehicle Control	1.98	0.85
Test Item 500 mg/kg	2.55	1.03
Test Item 1000 mg/kg	1.84	0.72
Test Item 2000 mg/kg	1.80	0.42
EMS 200 mg/kg	82.18	2.00

 

Table 3: Overview Tail Intensity in glandular stomach Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	3.88	1.28
Test Item 500 mg/kg	4.78	1.33
Test Item 1000 mg/kg	5.83	2.06
Test Item 2000 mg/kg	4.72	1.23
EMS 200 mg/kg	54.99	3.96

 

Table 4: Overview Tail Intensity in duodenum Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	4.49	0.72
Test Item 500 mg/kg	3.94	0.66
Test Item 1000 mg/kg	3.74	0.54
Test Item 2000 mg/kg	4.06	0.80
EMS 200 mg/kg	40.43	6.07

Conclusions:

Under the study conditions, Propionaldehyde, reaction product with formaldehyde (EC No 701-281-9 / CAS No -) was shown to be not genotoxic in vivo in the comet assay in liver, glandular stomach and duodenum cells, 3-4h post gavage exposure of male Wistar rats up to a dose of 2000 mg/kg bw/day. The validity of the test was confirmed.

Executive summary:

A study was conducted to evaluate the potential in vivo genotoxicity of Propionaldehyde, reaction product with formaldehyde (EC No 701-281-9 / CAS No -) according to OECD Guideline 489, in compliance with GLP. The test substance was administered by oral gavage to male Han Wistar rats up to the maximum recommended dose for two consecutive days and subsequent measurements of the increase in DNA strand breaks in liver, glandular stomach and duodenum were conducted when sampled approximately 3-4 hours post dosing.

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.

The study procedures described in this report were based on the most recent OECD guideline adopted on 29^thJuly 2016. Batch256710 001of the test item was a colourless liquid. The test item was dissolved in Milli-Q water. Based on the results of the dose-range finding study, test concentrations of 2000 mg/kg bw/day for male animals was selected as maximum dose for the main test (the highest dose required in the current guideline). Since there were no substantial differences in toxicity between sexes only males were used in the main study.

The concentrations analysed in the formulations of all groups were in agreement with target concentrations. No test item was detected in the vehicle formulations. The formulations were homogeneous. Formulations were stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.

In the main study, animals were dosed with vehicle (Milli-Q water)or test item (at 500, 1000 and 2000 mg/kg bw/day) for two consecutive days by oral gavage. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg bw/day.No clinical signs were observed in all animals.

Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated. Single cell suspensions from were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed.

No statistically significant increase in the mean Tail Intensity (%) was observed in liver, glandular stomach and duodenum cells of test item treated male treated animals compared to the vehicle treated animals.

The mean Tail Intensity in liver, glandular stomach and duodenum cells of vehicle-treated rats was 2.0±0.85% (mean± SD), 3.9±1.3% (mean± SD) and 4.5±0.72% (mean± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a statistically significant increase and showed a mean Tail Intensity of 82±2.0% (mean± SD), 55±4.0% (mean± SD) and 40±6.1% (mean± SD) in male animals in liver, glandular stomach and duodenum cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analysed (except for the liver which did not impact the study outcome) and the highest test dose was the maximum dose required by the guidelines. Hence, all criteria for an acceptable assay were met.

No test item-related changes in clinical chemistry parameters were noted in any of the test item treated groups. A slightly lower aspartate aminotransferase (ASAT) activity was observed in males at 500, 1000 and 2000 mg/kg bw/day. In absence of a dose-related response and at the magnitude of change, this finding was considered to be not toxicologically relevant. Furthermore, a slight increase in creatinine concentration was noted in males at 1000 mg/kg bw/day. In absence of a dose-related response, this finding was considered to be not test item-related. In conclusion, no relevant test item-related clinical chemistry changes were observed in males up to 2000 mg/kg bw/day.

Under the study conditions, Propionaldehyde, reaction product with formaldehyde (EC No 701-281-9 / CAS No -) was shown to be not genotoxic in vivo in the Comet assay in liver, glandular stomach and duodenum cells. The validity of the test was confirmed.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 14 June 2013 to 26 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 165.2 - 182.7 g
- Assigned to test groups randomly: yes, under following basis: proportionally to body weight
- Fasting period before study: no
- Housing: 5 animals per macrolon cages (type IV) with wood shavings (Lignocel, type ¾) as bedding material and a wooden block and strips of paper as environmental enrichment (Enviro-dri)
- Diet: cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3; pelleted) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum
- Water: domestic mains tap water suitable for human consumption, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 10

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water for injection
- Amount of vehicle: 10 ml/kg bw
- Lot/batch no.: 11054402

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was freshly formulated in water for injection. The exact volume of the dose formulation was calculated for each animal individually based on body weight.

Duration of treatment / exposure:

2 days

Frequency of treatment:

once daily

Post exposure period:

24 hours

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C (MMC)
- Route of administration: intraperitoneal
- Doses / concentrations: 1.5 mg/kg bw

Tissues and cell types examined:

Bone marrow: polychromatic and normochromatic erythrocytes

Details of tissue and slide preparation:

BONE MARROW COLLECTION AND PROCESSING
Immediately following sacrifice, the bone marrow cells of one of the femurs was collected into foetal calf serum and processed into glassdrawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were made, air-dried and fixed in methanol. One fixed smear was stained with a May-Grünwald Giemsa solution. The other fixed smear was kept in reserve and discarded after analysis of the stained smear.

Evaluation criteria:

The following criteria were used for the scoring of cells:
- A polychromatic erythrocyte (PE) is an immature erythrocyte that still contains ribosomes and can be distinguished from mature, normochromatic erythrocytes by a faint blue stain.
- A normochromatic erythrocyte (NE) is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by a yellow stain.
- A micronucleus is a small, normally round, nucleus with a diameter of circa 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain.

The numbers of PE and NE were recorded in a total of at least 200 erythrocytes (E) per animal. If micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total of 200 E (PE + NE) were scored, an additional number of PE were scored for the presence of micronuclei until a total of 2000 PE were scored.

The study was considered valid if the positive controls showed a statistically significant increase in the mean number of MPE/2000 PE and the vehicle controls
where within the historical range.
A test substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus if it showed a positive response, i.e. if the mean number of MPE/2000 PE was statistically significant higher compared to the vehicle control group.
The test substance or its metabolites were considered to be cytotoxic to the bone marrow via general circulation, if the test substance statistically significantly
reduced the mean number of PE.

Statistics:

Two ANOVA models were applied for both PE/200E and MPE/2000PE. The first ANOVA model tested if the positive control differed from the vehicle control (t test). The second ANOVA model tested if the vehicle control differed from the test substance. If the ANOVA assumptions were not valid (i.e. if variances were not
equal) non-parametric testing was performed and Mann Whitney p-values were reported. In all statistical tests a significance level of 5% was used (α = 0.05). All statistical tests were performed using GraphPad Prism®, Version 5.03, Copyright © 1992-2010 GraphPad Software, Inc., CA, USA.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No abnormalities were observed in any of the animals either ca. 1 h, 4 h or 24 h after each dosing

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no
- Ratio of PE/200E: 109 ± 11
- Appropriateness of dose levels and route: Indications of systemic availability of the test substance were obtained in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (induction of liver enzymes was observed), demonstrating that the negative response observed in this bone marrow micronucleus test is not due to lack of systemic availability of the test substance or its metabolites.

Conclusions:

The test substance Dimethylopropanal did not show any indication of chromosomal damage and/or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats, when orally dosed on two consecutive days with approximately 24 h interval, up to the limit dose of 2000 mg/kg bw.

Executive summary:

The test substance Dimethylolpropanal was examined for its potential to cause damage to the chromosomes and/or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow of male rats according to OECD Guideline 474 and under GLP.

Prior to the bone marrow micronucleus test, a dose confirmation study was performed in rats to determine tolerability of the test substance at the limit dose level of 2000 mg/kg-bw, in combination with the dosing regimen used in the current study. Two male rats were dosed twice orally (by gavage) on two consecutive days with approximately 24 h interval, with a dose level of 2000 mg/kg-bw Dimethylolpropanal (dosing volume 10 ml/kg-bw). No abnormalities were observed in either of the animals ca. 1 h, 4 h or 24 h after each dosing. Based on these observations a dose level of 2000 mg/kg-bw Dimethylolpropanal was considered suitable for the bone marrow micronucleus test.

In the bone marrow micronucleus test, rats were dosed twice orally (by gavage), on two consequentive days with approximately 24 h interval, with 2000 mg/kg-bw of the test substance or water for injection (vehicle control). Rats of the positive control group were injected once intraperitoneally with 1.5 mg/kg-bw mitomycin C. The dosing volume was 10 ml/kg-bw for all treatment groups. Prior to each dosing the rats were weighed and the dosing volume was adjusted to the body weight. Five rats per treatment group were used.

No abnormalities were observed in any of the groups and no mortality occurred during the study.

Approximately 24 h after the final treatment, rats were sacrificed by decapitation whilst under CO2/O2 anaesthesia, and bone marrow cells were collected from one of the femurs and processed into smears for microscopic examination. The number of polychromatic erythrocytes (PE) per 200 erythrocytes (E) and the number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) were counted for each rat.

The incidence of MPE per 2000 PE, found in the positive control group, was statistically significantly higher compared to that of the vehicle control group (p<0.01). The incidence of MPE per 2000 PE in the vehicle control group was within the range of historical control data. Therefore, the study was considered valid.

The incidence of MPE per 2000 PE in the rats treated with 2000 mg/kg-bw Dimethylolpropanal showed no statistically significant difference from that observed in the vehicle control group.

The incidence of PE per 200 E in the rats treated with either 2000 mg/kg-bw Dimethylolpropanal or the positive control substance MMC showed no statistically significant difference from that found in the vehicle control group, which reflects a lack of toxic effects of these substances on erythropoiesis.

Under the conditions used in this study it is concluded that the test substance Dimethylolpropanal did not show any indication of chromosomal damage and/or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats, when orally dosed on two consecutive days with approximately 24 h interval, up to the limit dose of 2000 mg/kg-bw.

Indications of systemic availability of the test substance were obtained in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (induction of liver enzymes was observed), demonstrating that the negative response observed in this bone marrow micronucleus test is not due to lack of systemic availability of the test substance or its metabolites.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Two in vitro genotoxicity test were performed, covering the endpoints gene mutation in bacteria and chromosome aberrations in mammalian cells. Both tests were positive.

Two in vivo genotoxicity test were performed, covering the endpoint cytogenicity and cell repair in mammalian cells. Both tests were negative.

Justification for classification or non-classification

As justified with the two negative in vivo genotoxicity tests covering the test item does not need to be classified for genetic toxicity according to Regulation (EC) No 1272/2008.