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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
No evidence of mutagenicity was seen in an Ames test. A weak positive response is reported in a mouse lymphoma assay in the presence of metabolic activation and at only concentrations associated with marked cytotoxicity. Analysis of colony size in this study indicates that findings are due to clastogenicity rather than mutagenicity. No evidence of clastogenicity was seen in a standard mouse micronucleus assay, therefore it is concluded that trimethylolpropane diallyl ether (TMPDE) is not clastogenic in vivo.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 November to 10 December 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP and guideline-compliant study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Bor: NMRI (SPF Han)
Sex:
male/female
Details on test animals and environmental conditions:
The animals were young adult male and virgin female mice, strain Bor: NMRI (SPF Han), bred and supplied by F. Winkelmann, Borcehn. They initially weighed 28-41 g, and were approximately 8-12 weeks of age.

The breed's state of health was regularly spot checked for the major specific pathogens. On the day of arrival, the health of the animals was checked before acclimatising them to the housing conditions for a period of at least one week. Only healthy animals were used in the study.

The females were housed in groups of up to 3 in Makrolon type I cages. Males were singly housed in type I cages. Bedding of soft wood granules (spot-checked for contaminants at regular intervals), type S 8/15 (J. Rettenmaier & Sohne) was used. Individuals were identified by picric acid marks.
The room temperature was maintained at 22-23°C, and 46-52% mean relative humidity, artificial light was provided for 12 hours per day, and there were approximately 10 air changes per hour.

Tap water and feed (Altromin 1324 Standard Diet) were provided ad libitum. The nutritive composition and contaminant content of the diet were checked regularly, along with water quality.
Mice were randomly divided into treatment groups by a randomisation plan, produced by the Insititue of Biometrics, Bayer AG.
Route of administration:
intraperitoneal
Vehicle:
Corn oil
Details on exposure:
The test substance was dissolved in corn oil and injected i.p. The injection volume was 5 ml/kg bw.
Duration of treatment / exposure:
Mice were sacrificed at 16, 24 or 48 hours following adminsitration.
Frequency of treatment:
Single i.p. injection.
Post exposure period:
A post exposure period was not included, mice were sacrificed at varying times following administration.
Remarks:
Doses / Concentrations:
1250 mg/kg bw
Basis:
no data
No. of animals per sex per dose:
5 mice/sex/dose
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
The positive control was Cyclophosphamide, in the form of Endoxan 100 mg injection vials of dry sbstance (Asta), batch 091520. Cyclophosphamide was dissolved in deionised water and injected i.p. The dose was 20 mg/kg bw, and the injection volume was 10 ml/kg bw.
Tissues and cell types examined:
Bone marrow - erythrocytes
Details of tissue and slide preparation:
Schmid's method was used to produce the smears. At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor. A tube was filled with foetal calf serum, a small amount of serum was drawn from the tube into a suitable syringe with a thin cannula. Te cannula was pushed into the open end of the marrow cavity of the femur. The femur was then completely immersed in the calf serum, the contents were then flushed several times and the bone marrow was passed in the serum as a fine suspension. If necessary the flushing was repeated from the other end of femur. The tube was then centrifuged at approximately 1000 rpm for 5 minutes. The supernatant was removed, leaving a small amount remaining. The sediment was mixed to produce a homogenous suspension. One drop of the viscous suspension was placed on a clean slide and spread. The labelled slides were dried overnight. If fresh smears needed to be stained, they were dried with heat for a short period.
The smears were stained automatically with an Ames Haematek Slide Stainer. The slides with then 'destained' with methanol, rinsed with deionised water, and left to dry.
Following this treatment, the smears were then transferred to a holder. A cuvette was filled with xylene, and the holder was immersed into the cuvette for approximately 10 minutes. The slides were removed singly and covered. A small amount of covering agent was taken from a bottle and applied to the coated side of the slide. A cover glass was then placed in position. Slides were not evaluated until the covering agent had dried.
Evaluation criteria:
Coded slides were evaluated using a light micropscope. 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. The number of normochromatic erythrocytes showing micronuclei was also established.
A test was considered positive in there was a relevant and significant increase in the number of micronucleated polychromatic erythrocytes compared to the negative control.
Statistics:
Wilcoxon's non-parametric rank sum test, and one-sided chi-square test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
There were no differences in the results between males and females, therefore both sexes were evaluated together. No biologically important or statistically significant variations existed between the negative control and treated groups, with respect to the incidence of micronucleated polychromatic erythrocytes, micronucleated normoerythrocytes. The ratio of polychromatic to normochromatic erythrocytes was altered, in a biologically relevant manner, in the treated groups compared to the negative control. The ratio was not altered in the positive controls. The positive control caused a clear increase in the number of micronucleated polychromatic erythrocytes.

After a single i.p. dose of 1250 mg/kg bw, treated mice showed apathy, anaesthethised-like state, roughened fur, staggering gait, sternal recumbency, spasm, shivering, difficulting breathing and breathing noises. Symptoms were present until sacrifice. Feeding behaviour was normal (no data included to support this). There were no mortalities. The number of micronucleated PCEs was comparable in all treated groups and control: an appropriate response was seen to the postive control compound (CPA).

Table 1. Summary of Results

Treatment

Sacrifice (hours after treatment)

No. evaluated polychromatic erythrocytes (n=10 mice)

No. normochromatic erythrocytes per 1000 polychromatic erythrocytes

Micronucleated cells per 1000

Normochromatic erythrocytes

Polychromatic erythrocytes

Negative Control (corn oil)

24

10000

573 ± 143

1.2 ± 1.1

1.7 ± 1.5

TMPDE

16

10000

1207 ± 402

1.2 ± 1.0

2.6 ± 2.2

24

10000

790 ± 245

1.6 ± 2.1

1.7 ±1.4

48

10000

782 ± 186

1.5 ± 0.9

1.5 ± 1.2

Positive Control (cyclophosphamide)

24

10000

483 ± 135

1.6 ± 2.1

17.9 ± 6.6*

Data shown are mean ± 1 standard deviation

TMPDE = trimethylolpropandiallylether (1250 mg/kg bw i.p.)

* P < 0.01

Conclusions:
Interpretation of results (migrated information): negative
There was no evidence of a clastogenic effect following a single i.p. administration of 1250 mg/kg bw in mice.
Executive summary:

A mouse micronucleus test was employed to assess the clastogenic potential of trimethylolpropandiallylether, in male and female mice, according to OECD method 474. The mice received a single intraperitoneal injection of the test substance at a dose level of 1250 mg/kg bw in corn oil. Vehicle controls and positive controls (cyclophosphamide) were included. The treated mice were sacrificed at 16, 24 and 48 hours after administration. All treated mice survived to sacrifice, however all showed signs of toxicity. There was an altered ratio between polychromatic and normochromatic erythrocytes. No evidence of a clastogenic effect was found. The response to the posive control (CPA) confirmed the sensitivity of the assay.

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

Additional information

Additional information from genetic toxicity in vivo:

Mutagenicity

No evidence of bacterial reverse mutation was seen in a modern, guideline-compliant Ames test using strains of S. typhimurium and E. coli (Wright, 1993); the study is clearly negative.

In a modern guideline-compliant mouse lymphoma assay (Riach, 2010) no evidence of mutagenicity was seen in the absence of metabolic activation. Weak responses (meeting the laboratory's criteria for a postive response) were seen in the presence of metabolic activation at higher concentrations. Positive responses were associated with marked cytotoxicity (relative total growth of 8 -15%). The report authors note a preponderance of small type colonies at these concentrations, indicating that the activity of TMPDE in this study is associated with large-scale (i.e.chromosomal) damage rather than deletions and/or point mutations. The authors further conclude that, while this result is insufficient evidence on its own to classify the substance as mutagenic according to GHS Category 2, it would contribute towards a Category 2 classification if supported by positive findings from relevant in vivo tests.

A negative result was achieved in an Ames test conducted by Bayer AG Leverkusen, using strains of S. typhimurium. Bacteritoxic effects were seen at all doses used (up to 12500 µg/plate). No indications of mutagenic effects were found at evaluable doses of up to 4800 µg/plate. Access to the original study report is not available, the result was obtained from the IUCLID data set (2000) and is presented in support of the data summarised above.

Clastogenicity

The results of the mouse lymphoma assay (Riach, 2010) indicate that TMPDE is clastogenic in vitro, in the presence of metabolic actviation and at concentrations associated with marked cytotoxicity. Further studies of chromosomal aberration in vitro are not available, however the substance has been investigated in a higher tier (in vivo) study.

TMPDE was investigated in a micronucleus test (Herbold, 1993) in male and female mice, according to OECD method 474. The mice received a single intraperitoneal injection of the test substance at a dose level of 1250 mg/kg bw in corn oil. Vehicle controls and positive controls (cyclophosphamide) were included. The treated mice were sacrificed at 16, 24 and 48 hours after administration. All treated mice survived to sacrifice, however all showed signs of toxicity. There was an altered ratio between polychromatic and normochromatic erythrocytes. No evidence of a clastogenic effect was found in this study. The response to the posive control (CPA) confirmed the sensitivity of the assay.

The negative findings in the mouse micronucleus study therefore indicate that TMPDE is not clastogenic in vivo.


Justification for selection of genetic toxicity endpoint
Higher tier study

Justification for classification or non-classification

No classification is proposed according to EEC Council Directive 79/831/EEC Annex VI, Part II (D) as described in Commission Directive 93/21/EEC or Regulation (EC) No 1272/2008. No evidence of mutagenicity was seen in an Ames test. A weak positive response is reported in a mouse lymphoma assay in the presence of metabolic activation and at only concentrations associated with marked cytotoxicity. Analysis of colony size in this study indicates that findings are due to clastogenicity rather than mutagenicity. No evidence of clastogenicity was seen in a standard mouse micronucleus assay, therefore it is concluded that trimethylolpropane diallyl ether is not clastogenic in vivo.