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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Propionaldehyde, reaction products with formaldehyde
EC Number:
701-281-9
Molecular formula:
C5H10O3
IUPAC Name:
Propionaldehyde, reaction products with formaldehyde
Details on test material:
- Name of the test item as cited in report: Propionaldehyde, reaction product with formaldehyde
- Batch Number: 256710/01
- Expiration Date: May 05, 2022
- Storage Conditions: Room Temperature approx. 20 °C, not above 40 °C.
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

Test animals

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%.

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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 29thJuly 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.

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