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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data from in vitro studies on the test substance were considered ambigous. Effects occured at cytotoxic levels or were not reproducible. The hydrolysis product ethylene glycol was considered negative. Data on formaldehyde are presented as additional information.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
permanent V79 cell line UI/109 from the Chinese hamster.
Forward mutation assay using the HPRT gene; cells deficient in HPRT (due to mutation) are selected by medium containing 6-thioguanine.
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Without MA: 2, 5, 10, 15, 20, 25 µg/ml (3 h exposure); 2, 5, 10, 15, 20, 25 µg/ml (6 h exposure); 1, 2, 5, 7.5, 10, 12.5, 15 µg/ml (repetition 6 h exposure).
With MA: 1, 2, 5, 10, 20, 40 µg/ml (3 h exposure); 0.25, 0.5, 1, 2, 5, 10, 20, 40 µg/ml (6 h exposure)
Vehicle / solvent:
nutrient medium (DMEM)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Positive control:
1 µl/ml ethylmethane sulfonate without metabolic activation (MA)
10 µg/ml dimethylbenz(a)anthracene with MA
Evaluation criteria:
as described in OECD guideline 476
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Weak mutagenic activity with and without metabolic activation at concentrations near the cytotoxic range.
Executive summary:

Study performed according to the OECD guideline 476. V79 cells were exposed without metabolic activation (MA) to: 2, 5, 10, 15, 20, 25 µg/ml (3 h exposure); 2, 5, 10, 15, 20, 25 µg/ml (6 h exposure); 1, 2, 5, 7.5, 10, 12.5, 15 µg/ml (repetition 6 h exposure). With MA the following concentrations were used: 1, 2, 5, 10, 20, 40 µg/ml (3 h exposure); 0.25, 0.5, 1, 2, 5, 10, 20, 40 µg/ml (6 h exposure) The test substance induced an increased number of mutant cells with and without metabolic activation. The test substance was considered to be mutagenic under the condition of this study. Although study results might be discussed critically due to the fact that mutagenic effects occurred at concentrations near cytotoxic concentrations. Results are presumably also due to the hydrolysis product formaldehyde. Weak mutagenic activity with and without metabolic activation at concentrations near the cytotoxic range.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix; i.p. injection of phenobarbital + ß-naphtoflavone (80mg/kg body weight ) 5 days prior to sacrifice
Test concentrations with justification for top dose:
Pre-experiment on cytotoxicity: 0.2-100 µg/ml with (3 h of incubation) and without (24 h of incubation) metabolic activation (MA).
Main study: Mitotic index (M.I.): 5, 10, 15, and 20 µg/ml with and without MA (3 h of incubation); after evaluation of the M.I. 3 concentrations were chosen for analysis of chromosome aberrations. Analysis of chromosome aberrations: 5, 15, and 20 µg/ml with and without MA (3 h of incubation).
Vehicle / solvent:
culture medium (DMEM)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Evaluation criteria:
Pre-experiment on cytotoxicity: M.I.: 1000 cells evaluated per culture (number of cells in division/total number of cells)
Main study: Duplicate cultures for each concentration, concurrent negative (vehicle culture medium) and positive controls (EMS, CP); 3 h of incubation with and without MA; change to fresh culture medium, addition of 0.4 µg/ml Colcemid® per culture 21 h after treatment start, fixation 24 h after treatment start;
analysis of: M.I.: 1000 cells evaluated per culture (number of cells in division/total number of cells)
Chromosomal aberrations: 100 metaphases per culture, 200 metaphases per concentration; specification of aberration types in the results section, no statistical analysis performed
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
po
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Without metabolic activation: Concentration-dependent increase in chromosome aberrations; positive at 15 and 20 µg/ml (see Table below). Valid positive control. Negative control within the historical range concerning aberrations excluding gaps.

With metabolic activation: Concentration-dependent increase in chromosome aberrations; only positive at 20 µg/ml (see Table below). Valid positive control.

Cytotoxicity:

Without S9 mix: cytotoxicity at 15 and 20 µg/ml, mean reduction in M.I. to 64.9 and 51.6% of the respective negative controls

With S9 mix: cytotoxicity at 20 µg/ml, mean reduction in M.I. to 49.9% of the respective negative controls

Conclusions:
[1,2-ethanediylbis(oxy)]-bis-methanol is considered to be clastogenic in V79 cells at concentrations of cytotoxicity.
Executive summary:

In a chromosome aberration test according to OECD 473 and GLP V79 Chinese hamster lung fibroblasts were cultured for 3 h with 0, 5, 15, and 20 µg test substance/ml with and without metabolic activation. Under the test conditions described above, the test item showed a dose-dependent increase in the number of cells with chromosome aberrations both with and without MA tested in the dose-range of 5-20 µg/ml. Aberrations mainly consisted of breaks and exchanges (mainly chromatid-type). Induction of aberrations was less intense in the presence of S9 mix, pointing to metabolic detoxification of the test item. [1,2-ethanediylbis(oxy)]-bis-methanol is considered to be clastogenic in V79 cells at concentrations of cytotoxicity.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
TA1537 His C 3076; rfa-; uvrB frame shift mutations
TA98 his D 3052; rfa-; uvrB-; R-factor frame shift mutations
TA1535 His G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-; R-factor base-pair substitutions
E. coli WP2 uvrA trp-; uvrA-: base-pair substitutions and others.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The Reaction Product formed between Ethylene glycol and Paraformaldehyde was dissolved in dimethyl sulfoxide and administered at the following levels:
Exp. I Rangefinding: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate;
Exp. II Mutation assay: 1, 3, 10, 33, 100 and 333 μg/plate;
Exp. III Mutation assay: 1, 3, 10, 33, 100 and 333 μg/plate (strains TA 98 and TA 100 only).
Positive controls: sodium azide: 10.0 µg/plate;
4 nitro-o-phenylene-diamine: 10.0 and 50.0 µg/plate;
Methyl methane sulfonate: 5.0 µL/plate;
2 aminoanthracene: 2.5 and 10.0 µg/plate.
Negative control: no treatment.
Solvent control: dimethyl sulfoxide.
Bacterial culture: 0.1 mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4 nitro-o-phenylene-diamine, 2-aminoanthracene
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or three times (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Without metabolic activation

Range finding (Exp I): The test material was toxic to TA 1535, TA 1537, and TA 98 at 333 to 5000 µg/plate and to TA 100 and WP2 uvrA at 1000 to 5000 µg/plate. 

Mutation assay (Exp II): Solvent controls gave normal counts of spontaneous revertants. Positive controls gave expected counts of induced revertants. Test material was toxic to TA 1535,TA 100, and WP2 uvrA at 333µg/plate, to TA 1537 at 33 to 333 µg/plate, and to TA 98 at 100 to 333 µg/plate. 

None of the bacterial strains exposed to test material showed a doubling of revertants compared to controls. No dose-response relationship was observed for any strain. 

Appropriate reference mutagens were used as positive controls, and showed a distinct increase of induced revertant colonies. 

 

With metabolic activation:      

Rangefinding (Exp I): The test material was toxic to all strains at 1000 to 5000 µg/plate. 

Mutation assay (Exp II): Test material was toxic to all strains at 333 µg/plate. 

Mutation assay (Exp III): Test material was toxic to TA 98 and TA 100 at 333 µg/plate. 

In Exp. I, no substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level. 

In Exp. II, an increase in revertant colony numbers was observed in strains TA 98 and TA 100 in the presence of metabolic activation. The threshold of two times the corresponding solvent control was exceeded in strain TA 98 at 33 and 100 μg/plate. In strain TA 100 the threshold of twice the corresponding solvent control was not reached. However, an increase in revertant colony numbers was observed at 33 and 100 μg/plate. 

In Exp. III, a slight increase in revertant colony numbers was obtained in strain TA 100 at 100 μg/plate. However, the threshold of twice the corresponding solvent control was again not reached and the effects of both experiments are likely to be based on low colony counts of the corresponding solvent controls as compared to the corresponding negative control. In strain TA 98 the effects obtained in Exp. II could not be reproduced although identical concentrations were used. Only a slight increase was obtained at 33 and 100 μg/plate. Although the test results do not indicate a clear mutagenic potential, a mutagenic effect cannot be entirely excluded for strains TA 98 and TA 100 in the presence of metabolic activation. 

Appropriate reference mutagens were used as positive controls, and showed a distinct increase of induced revertant colonies. 

 

Cytotoxicity    

A range finding examination was conducted to investigate cytotoxicity. Cytotoxicity was assessed during all experiments, as described above. 

Conclusions:
During the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used with the exception of strains TA 98 and TA 100. Therefore, a possible mutagenic potential cannot be entirely excluded.
Executive summary:

The study was conducted under GLP with no protocol deviations.  During the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used with the exception of strains TA 98 and TA 100. Therefore, a possible mutagenic potential cannot be entirely excluded. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In a study according to OECD guideline 474 and GLP with 5 male + 5 female NMRI mice per dose and sampling time the test substance did not induce a significant increase in the number of micronuclei at dose levels up to 300 mg/kg b.w. in both m and f, with 300 mg/kg b.w. mediating marked toxicity. The ratio of PCE / NCE was also not significantly altered by treatment of animals with the test substance. There are valid positive and vehicle controls. The negative controls lie within in the range of historical negative controls. Under the restrictions of the assay the test substance is not considered to be mutagenic in PCE of the bone marrow of NMRI mice.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
Charles River Deutschland GmbH, 97633 Sulzfeld,
Weight at study initiation: m: 23 days (at start of adaptation) / 22-26 g (at administration), f: 24 days (at start of adaptation) / 20-25 g (at administration)
Photoperiod (hrs dark / hrs light): 12/12;
Housing: 5 animals per cage, identification by coloured marks and cage labels;
Temperature (°C):22°C ± 3°C;
Humidity (%): 55% ± 15% relative humidity;
Food and water: ad libitum, feeding was discontinued approx. 16h before administration of the test item
Route of administration:
oral: gavage
Vehicle:
corn oil
Duration of treatment / exposure:
Single oral application
Frequency of treatment:
Single oral application
Post exposure period:
24 h (all dose levels including vehicle and positive controls) and 48 h (all dose levels except the positive controls) after treatment.
Remarks:
Doses / Concentrations:
0, 30, 100 and 300 mg/kg bw
Basis:
nominal conc.
Test substance concentration in vehicle: 0, 1.5, 5 and 15 mg/ml (0, 0.15, 0.5, and 1.5%)
No. of animals per sex per dose:
5 m + 5 f per dose and sampling time
Control animals:
yes
Positive control(s):
Vehicle control: Corn oil 20 ml/kg b.w. (oral, by gavage)
Positive control: cyclophosphamide, 27 mg/kg b.w. (i.p., 20 ml/kg b.w.)
Tissues and cell types examined:
Bone marrow; Erythrocytes
1000 red blood cells (RBC) for the ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE)
2000 PCE for the incidence of micronuclei
Evaluation criteria:
PCE / NCE ratio
Number of micronuclei in PCE per 1000 RBC
Statistics:
Chi-square test corrected for continuity according to Yates.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
No signs of toxicity at 30 and 100 mg/kg b.w.; Reduced motility, ataxia and dyspnoea at 300 mg/kg b.w. (30-60 min after administration)
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid

Preliminary dose-range-finding study: Toxic effects at 250 mg/kg b.w. (reduced motility, ataxia and dyspnoea) more pronounced at 500 mg/kg b.w.; 1000 mg/kg b.w. was lethal Main study: No signs of toxicity at 30 and 100 mg/kg b.w.; Reduced motility, ataxia and dyspnoea at 300 mg/kg b.w. (30-60 min after administration).

The PCE / NCE ratio was not significantly altered by treatment with the test item, compared to the concurrent vehicle controls. The ratios were within the range of the historical vehicle control data of the same laboratory

Conclusions:
Under the restrictions of the assay the test substance is not considered to be mutagenic in PCE of the bone marrow of NMRI mice. No classification is indicated a according to Regulation (EC) No 1272/2008.
Executive summary:

In a study according to OECD guideline 474 and GLP 5 male + 5 female NMRI mice per dose and sampling time were used; mice received a single oral application via gavage at dose levels of 0, 30, 100 and 300 mg/kg bw (0, 0.15, 0.5, and 1.5% in corn oil). 24 h (all dose levels including vehicle and positive controls) and 48 h (all dose levels except the positive controls) after treatment mice were sacrificed and bone marrow prepared. There are valid positive and vehicle controls. Cyclophosphamide as positive control induced a significant increase in micronucleus formation in both m and f. The negative controls lie within in the range of historical negative controls. Under the conditions of this assay the test substance did not induce a significant increase in the number of micronuclei at dose levels up to 300 mg/kg b.w. in both m and f, with 300 mg/kg b.w. mediating marked toxicity. The ratio of PCE / NCE was also not significantly altered by treatment of animals with the test substance.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Data on formaldehyde

Genotoxicity in vitro

In the Salmonella microsome assays gene mutations were induced with and without metabolic activation. In mammalian cells, chromosome aberrations were detected independent of the metabolic activation system. There is also evidence for gene mutation in mammalian cells without a metabolic activation system. The DNA-protein cross-linking activity which occurred in vivo at the site of first contact has also been demonstrated in in vitro experiments. In further studies (IARC 1995), formaldehyde induced DNA damage in bacteria, forward and reverse mutation in bacteria (S. typhimurium and E. coli), DNA damage in fungi including DNA-protein cross-links (DPC), gene conversion and gene mutation in fungi, and in mammalian cells DNA damage including DPC, sister chromatid exchange and cell transformation.

In summary, gene and chromosome mutagenic activity of formaldehyde are well documented from in vitro studies and numerous studies on other endpoints suggested further evidence for genotoxicity of formaldehyde in vitro.

Genotoxicity in vivo

Standard cytogenetic and micronucleus assays on systemic effects in samples of bone marrow or peripheral blood after oral or inhalation exposure in experimental animals revealed negative results. An oral rodent dominant lethal assay of limited validity was also negative, while i.p. studies (limited validity) gave positive results. Genotoxicity studies in Drosophila on different endpoints gave positive results after feeding of formaldehyde, however, the relevance for mammals is questionable. He et al.(1998) reported weak effects in the micronucleus assay in lymphocytes of students exposed during an anatomy class. Further, in a recent study by Orsiere et al. (2006) a significant increase in the incidence of monocentromeric micronuclei (indicating aneugenic effects) in peripheral lymphocytes of exposed pathologists/anatomists was reported, while no DNA damage including clastogenic effects were found. In other studies positive as well as negative results were reported. However, the evaluation of these studies is hampered by the low number of subjects, co-exposure and lack of details.

In conclusion, the available studies are not sufficient to draw a conclusion on possible systemic genotoxic effects of formaldehyde.

However, local clastogenic activity was found in studies with rats in the lung after inhalation (Dallas et al. 1992) or in the gastrointestinal tract after oral application (Migliore et al. 1989). A human cohort study on workers of a plywood factory revealed also evidence for slight local clastogenic effects in the nasal epithelium in coincidence with increased inflammation (Ballarian et al. 1992). Further, slight clastogenic effects but not aneugenic effects were shown in students exposed during an embalming course. These effects were more obvious in buccal cells than in cells of the nasal epithelium (Titenko-Holland et al. 1996). In a review presented by Speit & Schmid (2006) the local chromosome mutagenic effects of formaldehyde in humans after inhalation exposure in the micronucleus test with exfoliated nasal or buccal epithelial cell, the actual targets of formaldehyde were evaluated. The evaluation was based on 8 studies including the two most relevant studies cited above. The data suggest an increase in micronuclei frequencies in nasal and/or buccal cells after inhalation exposure. However, methodological shortcomings and limited documentation were found and according to the authors it is yet not possible to assess the local genotoxicity of formaldehyde in humans.

In several studies on rats and monkeys the formation of DNA-protein cross-links (DPC) at the site of first contact has been demonstrated after inhalation exposure. DPC formation was detected already at low concentrations (0.3 ppm in rats). The dose response was nonlinear with a steep rise at higher dose levels suggesting saturation of defence mechanisms and showed coincidence with damage of epithelium. Rapid removal of DPC has been reported in corresponding experiments (half-lives 2-4 h) and no accumulation was detected in in vivo studies.

In conclusion, the available data in experimental animals demonstrated the genotoxic activity of formaldehyde at the site of first contact after oral exposure. Studies on local mutagenic effects in humans suggested increased micronucleus frequencies in nasal and buccal cells, however, a final conclusion is not yet possible. The mechanism of clastogenicity might be related to DNA-protein cross-links and their repair. DNA-protein cross-links at the site of first contact have been demonstrated after inhalation exposure in rats and monkeys. There is no clear evidence for systemic genotoxicity in experimental animals. The data on systemic genotoxic effects in humans are conflicting.

Justification for classification or non-classification

Based on results of the in vivo study no classification is regarded necessary for the test substance according to Regulation (EC) No 1272/2008. Also for the hydrolysis product ethylene glycol no classification is regraded necessary.

However, the hydrolysis product formaldehyde has a harmonised classification as Muta. 2.