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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 2001-09-12 to 2001-11-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Version / remarks:
(1997)
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Chemical structure
Reference substance name:
Glyoxal
EC Number:
203-474-9
EC Name:
Glyoxal
Cas Number:
107-22-2
Molecular formula:
C2H2O2
IUPAC Name:
oxalaldehyde
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B 61
- Expiration date of the lot/batch:
- Purity test date: October 31, 2001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Glyoxal as 40% aqueous solution is stable for 6 months. The stability of the test substance as such and throughout the study period had been verified by reanalysis
- Solubility and stability of the test substance in the solvent/vehicle: the stability of the test substance (aqueous solution) at room temperature in the vehicle water also had been determined analytically in the course of the determination of the reanalysis

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd, Margate, UK
- Age and weight at study initiation:
Range-finding study: the males were about 7 weeks old whereas the females were about 8 to 9 weeks old. At test initiation the males weighed between 189 and 196 g and the females weighed between 152 and 179 g.
Main test: only males were used, they were about 7 weeks old and weighed between 196 and 231 g at test initiation.
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: housed in groups of no more than four animals in solid-floored cages
- Diet: special diet, ad libitum
- Water: public supply water, ad libitum
- Acclimation period:
Range-finding study: 1 to 7 days
Main test: 7 to 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 47 - 67
- Air changes (per hr): at least 15 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

OTHER
in order to enrich the environment and enhance the welfare of the animals, they were provided with wooden Aspen chew blocks .

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PRELIMINARY RANGE FINDER
A group of 3 male rats were dosed once with 2540 mg/kg bw Glyoxal and 2 groups of three female rats were dosed once with 1270 mg/kg bw or 2540 mg/kg bw of the test substance. Dosing consisted of a single application by gavage. The animals were observed for clinical toxicity, and on the basis of the results a maximum tolerable dose was selected for the main test.

MAIN ASSAY
- On the basis of the results of the range finding test, 2000 mg/kg bw was selected as maximum tolerable dose for male Wistar rats;
- The assay comprised two experiments with different post-treatment period: 12 –14 h and 2 – 4 h;
- The animals received single oral application of test solution at an application volume of 10 mL/kg bw;
- The test animals were examined for clinical signs of toxicity and the body weights were recorded;
- At the end of the respective post-treatment periods, the rats were sacrificed and the hepatocytes were isolated from the liver of each animal.
Duration of treatment / exposure:
Not applicable as single application
Frequency of treatment:
single administration by gavage
Post exposure period:
2-4 h or 12-14 h
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 other: mg/kg bw
Remarks:
Main assay; actual ingested
(the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)
Dose / conc.:
2 000 other: mg/kg bw
Remarks:
Main assay; actual ingested
(the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)
Dose / conc.:
1 270 other: mg/kg bw
Remarks:
Range-Finding test; actual ingested
(the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)
Dose / conc.:
2 540 other: mg/kg bw
Remarks:
Range-Finding test; actual ingested
(the doses referred to the active ingredient Glyoxal and were calculated starting from the 40% test substance using a density of 1.27 g/cm3)
No. of animals per sex per dose:
RANGE-FINDING
A group of 3 male rats were dosed once with 2540 mg/kg bw Glyoxal and 2 groups of three female rats were dosed once with 1270 mg/kg bw or 2540 mg/kg bw of the test substance.

MAIN TEST
A total of 32 males were used; each group comprised 4 animals.
Control animals:
yes
Positive control(s):
2-Acetamidofluorene (2-AAF, Sigma Chemical Co. Poole, UK) in corn oil, 75 mg/kg bw, was used for experiment 1.
Dimethylnitrosamine (DMN, Sigma Chemical Co. Poole, UK) in purified water, 10 mg/kg bw, was used for experiment 2.

Examinations

Tissues and cell types examined:
- At the end of the respective post-treatment periods, the rats were sacrificed and the hepatocytes were isolated from the liver of each rat;
- The cells were examined for cell viability as measured by the Trypan blue exclusion technique;
- DNA damage and repair was measured by incorporation of 3H-thymidine using autoradiography technique;
- For evaluation and quantification of UDS, a total of 100 cells/animal was examined and following parameters were considered: net nuclear grain (NNG) count/cell, group mean net nuclear grain (NNG) count, mean net grain (NG) count of cells in repair, percentage of cells in repair (cells with NNG >= 5).
Evaluation criteria:
- A positive response implicates a dose-related increase in mean number of NNG counts (> 0 at one of the test points) and in percentage of cells in repair (i.e. cells with NNG >= 5), which must be >= 20%.
- A negative response implicates that both, the NNG counts and the percentage of cells in repair are within the range of negative control.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
Glyoxal at 1000 and 2000 mg/kg bw resulted in NNG values < 0. In fact, group mean NNG values for the two experiments were in the range -0.8 to -2.6, and no more than 4.0% cells were seen in repair at any of the tested doses.
Toxicity:
no effects
Remarks:
In the main study one animal was found dead (first experiment, 2000 mg/kg dose level), which was surprising and not consistent with results of the range-finder experiment. No other clinical signs of toxicity were observed in any other main study animal.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
During the 2 day post-exposure period, the males treated with 2540 mg/kg bw showed weight loss whereas the females treated with the same dose were cold to the touch, pale, lethargic, with hunched posture and weight loss. One female animal was sacrificed in extremis whereas another one was found dead. No mortalities were seen in the group of females treated with 1270 mg/kg bw; the animals displayed piloerection, lethargy, abnormal breathing, protruding eyes and weight loss.

RESULTS OF DEFINITIVE STUDY
Cell viability in the first experiment at both dose levels (1000 and 2000 mg/kg bw) was quite similar and ranged between 55 and 67%; cell viability in both cases was within or close to negative (vehicle) control range (59 – 66%). Cell viability in the positive control group treated with 2-AAF also was within negative control range (55 – 67%).
Cell viability in the second experiment at both dose levels (1000 and 2000 mg/kg bw) was quite similar and ranged between 63 and 74%; cell viability in both cases was within or close to negative (vehicle) control range (69 – 78%). Cell viability in the positive control group treated with DMN also was within negative control range (70 – 77%).
For details on genotoxicity, see tables below:

Any other information on results incl. tables

Experiment 1, summary of findings referring to the group mean net grain count:

12 to 14 hour sacrifice time

Group

Dose (mg/kg bw)

Net grain count (NNG)

% of cells in repair (NNG ≥ 5)

Net grain count of cells in repair

Mean

SD

Mean

SD

Mean

SD

Neg. control

0

- 2.6

0.3

-

-

-

-

Glyoxal

1000

- 2.3

0.8

-

-

-

-

 

2000

- 2.6

0.8

-

-

-

-

2-AAF

75

26.5

11.5

99.0

1.0

26.7

11.3

Experiment 2, summary offindings referring to the group mean net grain count:

2 to 4 hour sacrifice time

Group

Dose (mg/kg bw)

Net grain count (NNG)

% of cells in repair (NNG ≥ 5)

Net grain count of cells in repair

Mean

SD

Mean

SD

Mean

SD

Neg. control

0

- 0.8

1.1

3.3

2.3

6.0

0.6

Glyoxal

1000

- 0.8

1.3

4.0

4.0

6.5

0.3

 

2000

- 1.3

1.5

3.3

2.1

6.1

0.7

DMN

10

25.4

1.4

92.7

2.5

27.2

1.7

Applicant's summary and conclusion