Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471 with 1,3 -BDDMA ongoing, Results expected in Q2/2020. Dossier will be updated without undue delay.

Mouse Lymphoma Assay, sim. OECD 476, GLP, +/- metabolic activation: positive only with S9, high cytotoxicity

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
The performance of the OECD 471/ Genetic toxicity in bacteria is in delay.
The (eco)toxicological endpoint requested by ECHA in the decision of October 2017 was initially addressed by read across to a similar substance, i.e. 1,4-BDDMA (submitted on 08.05.2014).
After the release of the Read Across Framework (RAAF) in 2017, defining obligations for read across under the REACh Regulation, an update of the read across approach was considered.
However, based on the new information on revised completeness check of REACH registrations presented by the ECHA officials C. Musset and A. Bichlmayr, respectively, in a recently performed workshop (VCI conference REACH & CLP, November 28, 2019 in Frankfurt, Germany), the need of additional data generation was identified in order to fulfil the requirements with higher suitability.
Commissioning of the tests took some time specifically since the contract laboratories were/are overwhelmed with requests for in vivo studies with similar deadlines as those of the Higher Methacrylate REACH Task Force. Our view is that notwithstanding the delay further time will be required to assess the result for 1-methyltrimethylene dimethacrylate and to allow the dossiers to be updated more adequately.
We expect to have the results of the OECD 471 by 30th June 2020 in a draft report. (Please refer to the attached Statement of ICCR-Rossdorf GmbH).
The REACH dossier will be updated without undue delay once the study finalised.
For this issue, please also refer to the "Letter to ECHA" as attached in IUCLID Section 13.2..
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix

Genetic toxicity in vivo

Description of key information

- Dom. Lethal Assay, pre-GLP, rat, oral feed: negative (Hess et al. 1981; read-across from the alcohol metabolite)

- Three gen. cytogenetic study, pre-GLP, rat, oral feed: negative (Hess et al. 1981; read-across from the alcohol metabolite)

- Dom. Lethal Assy, pre-GLP, rat, oral feed: negative (Anderson/Zeneca/ICI 1976; read-across from methacrylic metabolite)

- Cytogenetic study, pre-GLP, rat, oral feed: interpreted as negative (Anderson/Zeneca/ICI 1976; read-across from methacrylic metabolite)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
study with 3 generation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
Cytogenicity assay with three generations of a five generation study.
GLP compliance:
not specified
Type of assay:
other: Cytogenicity assay with bone marrow cells
Specific details on test material used for the study:
source: Celanese Chemical Company, New York
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: FDRL-stock
- Age at study initiation: (P) 14-15 wks
- Housing: individually
- Diet: semipurified diet, ad libitum
- Water: tap water, ad libitum
ENVIRONMENTAL CONDITIONS
controlled
- Temperature (°C): 22+-2
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test diets were prepared by substituting 1,3-butanediol for equal amounts by weight of corn starch and dextrose. No further information.
Duration of treatment / exposure:
F0 rats were treated 4 weeks before the mating period. Female rats of the F0 were fed diets containing 1,3-butanediol throughout the mating, gestation and lactating period.
Frequency of treatment:
daily
Dose / conc.:
5 other: %
Remarks:
nominal in diet
Dose / conc.:
10 other: %
Remarks:
nominal in diet
Dose / conc.:
24 other: %
Remarks:
nominal in diet
No. of animals per sex per dose:
At least two rats per sex per group were examined.
Control animals:
yes, plain diet
Positive control(s):
no
Tissues and cell types examined:
Animals from the FlA, F2A and F3A generations were sacrificed and bone marrow (femur) preparations were examined cytologically for aberrations in the chromosomal patterns associated with treatment.
Details of tissue and slide preparation:
The selected rats were injected intraperitoneally with colchicine (1 mg kg-1), 3-4 h prior to sacrifice. Following dissection, the marrow
was washed with 5 m1 of Hank's balanced salts solution. The cells were centrifuged, washed repeatedly with fresh Hank's solution and suspended in 6 ml of hypotonic fetal calf serum, which was incubated at 37°C for 20min. The cells were fixed in a 3 : 1 mixture of methanol-glacial acetic acid at 4 ° C overnight before being coated on coverslips and stained with 2% aceto-orcein. The preparations were
examined by phase-contrast microscopy at 900x magnification for aberrant chromosomes. One hundred to 250 metaphase
cells were examined per group.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Additional information on results:
The frequency of occurrence of abnormal cells was found to be within the normal range for FIA, F2A and F3A rats; thus, the specific aberrations are not given. No specific abnormalities were consistently observed in any diet group; no
Endpoint:
genetic toxicity in vivo, other
Remarks:
Dominant lethal assay
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
Dominant lethal assay with the F1B generation of a five generation study. Study protocol was basically in accordance with OECD 478.
GLP compliance:
not specified
Type of assay:
rodent dominant lethal assay
Specific details on test material used for the study:
source: Celanese Chemical Company, New York
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: FDRL-stock
- Age at study initiation: (P) 14-15 wks
- Housing: individually; during Dominant lethal assay in a mating cage
- Diet: semipurified diet, ad libitum
- Water: tap water, ad libitum
ENVIRONMENTAL CONDITIONS
controlled
- Temperature (°C): 22+-2
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test diets were prepared by substituting 1,3-butanediol for equal amounts by weight of corn starch and dextrose. No further information.
Duration of treatment / exposure:
F0 rats were treated 4 weeks before the mating period. Female rats of the F0 were fed diets containing 1,3-butanediol throughout the mating, gestation and lactating period. For the dominant lethal test involving F1B animals, ten males per group were reared to maturity.
Frequency of treatment:
daily
Dose / conc.:
5 other: %
Remarks:
nominal in diet
Dose / conc.:
10 other: %
Remarks:
nominal in diet
Dose / conc.:
24 other: %
Remarks:
nominal in diet
No. of animals per sex per dose:
10 males & in total 160 untreated females
Control animals:
yes, plain diet
Positive control(s):
no
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Additional information on results:
All mal es in all groups sired litters. The percentage of pregnancies as well as the percentage of viable fetuses per implant site were not significantly different between treatment and control groups. Also, the mutagenic index (resorptions as a percentage
of implant sites) showed no trend with increasing dosages of 1,3-butanediol in the diet.
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Method and results sufficient described, similar to OECD-guideline 478
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10-12 w
- Diet: Alderley Park mouse cubes
Route of administration:
inhalation
Details on exposure:
During exposure, male CD-1 mice were individually housed in chambers made of stainless steel and glass with an internal capacity of three liters. Seven groups of mice, previously shown to be fertile, were treated according to the scheme presented below.
Fertility testing: Prior to the five-day inhalation exposures, male mice were each mated with two virgin adult female mice for five days. After a five-day mating period, the females were transferred to other cages. The females were sacrificed 15 days following the first day of placement with the males and examined for pregnancy. Only males successful in mating were used on the test.
Experimental mating and necropsy: After treatment, male mice were individually housed. Two virgin female mice were placed in each cage. After a five-day mating period, the females were removed and pair-housed. After a two-day rest period, two new virgin female mice were housed with each male for a five-day mating period. This process was repeated until the male mice had been mated for eight weeks. The male mice were then sacrificed and discarded without necropsy. It was assumed the females were fertilized within two to three days after mating pairs were set up. Thirteen days after the fertilization date, each female was sacrificed and examined for pregnancy, living fetuses and early and late fetal development. 
Duration of treatment / exposure:
5 days, 6 hours/day
Frequency of treatment:
Daily
Dose / conc.:
0.405 mg/L air
Remarks:
corresponding to 100 ppm

Dose / conc.:
4.05 mg/L air
Remarks:
corresponding to 1000 ppm
Dose / conc.:
36.45 mg/L air
Remarks:
corresponding to 9000 ppm
No. of animals per sex per dose:
total number of animals: control: 35; test groups: 20; positive controls: 13, 5 and 12
Control animals:
yes, concurrent no treatment
Positive control(s):
200 mg cyclophosphamide in water/kg bw once by i.p. injection on day 5; 150 mg ethylmethane sulphonate in water/kg bw orally once a day for 5 days ; 2.5 mg meclorethamine in saline once 
Tissues and cell types examined:
1) total implants/pregnancy; early deaths/pregnancy; and early deaths/total implants/pregnancy. 
Statistics:
A simple 2X2 Chi-square was used to analyze the data. Also, a week-by-week hierarchical analysis of variance was applied. The following three responses on each female were analyzed: 1) total  implants/pregnancy; early deaths/pregnancy; and early deaths/total  implants/pregnancy. For response 2, the Freeman-Tukey Poisson variance stabilizing transformation was used. Non-pregnant females were taken as missing data. Dunnett's t-test was used for multiple comparisons.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mortality was observed in the three dose groups exposed to the test substance. One animal died in the 100-ppm group the week following exposure, one animal died (95% survival) in the 1000-ppm group and six animals died (70% survival rate) in the 9000-ppm group during exposure. Five animals from the cyclophosphamide positive control group died within eight weeks after dosing.  
Fertility Successful mating: No effects observed in the MMA-exposed groups. Positive controls showed appropriate reduction in fertility.
Pregnancy frequency: Reduction in the 1000-ppm group in week 6 only was not considered related to MMA toxicity. Positive controls showed a  decrease in frequency.
Total implantations: No effects observed in the MMA-exposed groups. Positive controls showed appropriate reduction in implant numbers.
Early deaths: Percentages of early deaths were not affected in the MMA-exposed groups. Positive controls showed an appropriate increase in the number of early deaths.
Mean number of early deaths: No effects observed in the MMA-exposed  groups. Positive controls showed an appropriate increase in the number of early deaths.
Percentage of total implantations per pregnancy that were early deaths: No effects observed in the MMA-exposed groups.  
Late deaths: No effects were observed in this study.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Method and results sufficient described, similar to OECD-guideline 475.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
no
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
other: Alderley Park
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Alderley Park
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 150-200 g
- Housing: individually
- Diet (e.g. ad libitum): Alderley Park rat cubes
- Water (e.g. ad libitum): unspecified
Route of administration:
inhalation
Vehicle:
unchanged
Duration of treatment / exposure:
single treatment: 2 hrs
repeated treatment: five hrs a day for 5 consecutive days
Frequency of treatment:
single treatment: once
repeated treatment: daily for 5 days
Post exposure period:
Animals were sacrificed 24 hr following the last exposure period.
Remarks:
Doses / Concentrations:
ca. 0.4, 4.1 and 36.9 mg/L (corresponding to 100, 1000 and 9000 ppm)
Basis:
nominal conc.
No. of animals per sex per dose:
single treatment: 2-4 rats per group or 5 rats per group
repeated treatment: 7 rats per group.
Control animals:
yes, concurrent no treatment
Positive control(s):
10, 750 and 7500 ppm benzene
Tissues and cell types examined:
bone marrow samples were collected and processed
Details of tissue and slide preparation:
Preparation according to the method of Sugiyama (1971) but with slight modifications.
Statistics:
The data were transformed using a variance stabilizing transformation; the data were analysed using an analysis of variance and a one sided students t test was used on the transformed data
Sex:
male
Genotoxicity:
ambiguous
Toxicity:
not specified
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The study was negative for chromosomal aberration frequencies when gaps were excluded as usual.
When the data from the two 2 hr exposure experiments were combined and gaps were included, 1000 and 9000 ppm MMA groups were significantly different from controls; there was evidence of a dose response relationship. The group exposed to 9000 ppm for five repeated exposures was significantly different from controls; there was evidence of a dose response relationship.

Combined abnormalities of the two single treatment assays

 

Treatment

% abnormal cells (out of 50cells)

Ctrl

5.1

100 ppm MMA

5.5

1000ppm MMA

8.0*

9000 ppm MMA

9.4**

10 ppm B

10.3**

750 ppm B

18.0***

7500 ppm B

26.5***

 B=Benzene

Abnormalities of the repeated treatment assay

 

Treatment

% abnormal cells (out of 50cells)

Ctrl

3.1

100 ppm MMA

1.4

1000ppm MMA

6.3

9000 ppm MMA

6.9*#

10 ppm B

7.4**

750 ppm B

10.0***

7500 ppm B

14.0***

 # Statistically significant, however, compared to the control in the single-treatment segment (5.1 % ab.) the increase is biologically unimportant and does not comprise a positive effect.

Mean % abnormalities (excluding gaps)

 

Treatment

Single treatment A

Single treatment B

Repeated treatment

Ctrl

1.0

1.2

0.3

100 ppm MMA

0.7

0.8

0.3

1000ppm MMA

3.3

2.0

0.3

9000 ppm MMA

1.0

2.0

1.2

10 ppm B

5.0

1.6

2.0*

750 ppm B

8.0*

4.0

2.0*

7500 ppm B

15.3**

11.6*

3.7*

 

*    = significance level: 5%

**  = significance level: 1%

*** = significance level: 0.1%

Conclusions:
Interpretation of results (migrated information): negative
No increase in chromosome aberrations excl. gaps. An allegedly significant increase in chromosome aberrations incl. gaps is due to an - in comparison - low control value and biologically unimportant compared to other control values. In addition, the biological significance of chromosome gaps is unclear and, as an isolated finding, would not be regarded as a positive test result.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

For the assessment of the mutagenic potential of 1,3-BDDMA several studies are available, which were conducted with methacrylic metabolite MMA, or the alcohol metabolite 1,3-BD.

Following data are available:

The OECD 471 with 1,3-BDDMA is ongoing and the results are expected on 30thJune 2020.

A Mouse Lymphoma Assay, similar to OECD 476, GLP (+/- metabolic activation) revealed positive effects only with S9 in parallel a high cytotoxicity was observed (Litton 1985).

 

For the methacrylic and the alcohol metabolite, in vivo data is available:

 

 


 

 

 

Although the structure of the alcohol metabolite, 1,3-BD, is not indicative for a structural concern, following information supports the assessment of the genotoxic potential of the parent ester 1,3-BDDMA: Along with a five generation reproductive toxicity study with 1,3-BD (Hess et al. 1981), the primary metabolite of 1,3-BDDMA, also dominant lethal and cytogenetic effects were studied in Wistar rats. Animals of both sexes were fed either control diet or diet supplemented with 1,3-butanediol at dose levels of 5, 10 or 24% of the diet by weight. For the dominant lethal assay of the Fl B generation, the mutagenic index (percentage resorptions per implant sites) revealed no dose-related trend. In the three-generation cytogenetic study, no 1,3-BD related chromosomal aberrations were noted.

 

For the other primary metabolite, also dominant lethal and cytogenetic effects were studied in CD-1 mice. Animal of both sexes had inhaled with MMA at doses of 100/ 1000 and 9000 ppm. For the dominant lethal assay, the mutagenic index (percentage resorptions per implant sites) revealed no dose-related trend. In the cytogenetic study, MMA induced chromosomal aberrations above 1000 ppm significantly different from the control. The results is interpreted as negative due to the low control value and biologically unimportant comparison to other control values. In addition, the biological significance of chromosome gaps is unclear and, as an isolated finding, would not be regarded as a positive test result.

 

Conclusion

This assessment is also in line with a recent peer-reviewed publication which assessed the genotoxic potential of the so called lower alkyl methacrylates (Albertini, 2017). Its summary reads as follows: “The genotoxicity of seven mono-functional alkyl esters of methacrylic acid… as well as methacrylic acid itself, the acyl component common to all, is reviewed and compared with the lack of carcinogenicity of methyl methacrylate, the representative member of the series so evaluated. Also reviewed are the similarity of structure, chemical and biological reactivity, metabolism and common metabolic products of this group of compounds which allows a category approach for assessing genotoxicity. As a class, the lower alkyl methacrylates are universally negative for gene mutations in prokaryotes but do exhibit high dose clastogenicity in mammalian cells in vitro. There is no convincing evidence that these compounds induce genotoxic effects in vivo in either submammalian or mammalian species. This dichotomy of effects can be explained by the potential genotoxic intermediates generated in vitro. This genotoxic profile of the lower alkyl methacrylates is consistent with the lack of carcinogenicity of methyl methacrylate.”

 

There are no data gaps for the endpoint genotoxicity. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

 

Compliance to REACh requirements

The in vitro mutagenicity test requirement for a gene mutation study is covered with a reliable Mouse Lymphoma assay, performed with the substance itself.

As already mentioned, the OECD 471 is going. The dossier will be updated without undue delay. The results are expected on 30thJune 2020.

All other mutagenicity test requirements are covered by read-across to studies the primary metabolites showing consistently negative in vivo results.

 

Reference:

Albertini, R.J. (2017): The lower alkyl methacrylates: Genotoxic profile of non-carcinogenic Compounds. Regulatory Toxicology and Pharmacology 84 77-93

Justification for classification or non-classification

Based on the available data 1,3-BDDMA is not considered to be mutagenic according to the criteria given in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC. Thus, no labelling is required.