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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Remarks:
screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 20 April to 25 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 422 (repeated dose and reproduction / developmental screening)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetramethylene dimethacrylate
EC Number:
218-218-1
EC Name:
Tetramethylene dimethacrylate
Cas Number:
2082-81-7
Molecular formula:
C12H18O4
IUPAC Name:
butane-1,4-diyl bis(2-methylacrylate)
Test material form:
liquid
Specific details on test material used for the study:
Identity : 1,4-Butanediol dimethacrylate
Alternative names : VISIOMER® 1.4 BDDMA
CAS number : 2082-81-7
EINECS number : 218-218-1
Appearance : Yellowish clear liquid
Appearance at first use : Colourless liquid
Storage conditions : Room temperature, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy S.p.A., Calco (Lecco), Italy

- Age at study initiation: (P) Males/females: approximately 8 - 9 wks
- Weight at study initiation: (P) Males: 196.5 - 204.7 g; Females: 166.1 - 189.3 g

- Fasting period before study:
- Housing: Pre mating period: no more than 5 per cage in clear polycarbonate cages measuring 59X39X20 cm with a stainless steel
mesh lid and floor (Techniplast - Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray will hold absorbent material which will be
inspected daily and changed at least three times a week.
During mating period: 1 male to one female per cage in clear polycarbonate cages measuring 36X19X24 cm with a stainless steel
mesh lid and floor (Techniplast - Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray will hold absorbent material which will be
inspected daily.
Pregnant females: will be transferred to individual cages after mating: solid bottomed, breeding cages (Techniplast - Gazzada S.a.r.l.,
Buguggiate, Varese), for the gestation period, birth and lactation.
Suitable nesting material will be provided and will be changed as necessary.
- Diet: ad libitum, commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4,
20019, Settimo Milanese (MI), Italy)
- Water: ad libitum, supplied via water bottles

- Acclimation period: aproximately 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2 °C
- Humidity (%): 55 ±15 %
- Air changes (per hr): 15 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume.

The required amount of 1,4-Butanediol dimethacrylate was suspended in the vehicle (corn oil) at the final concentrations of 20, 60 and 200 mg/mL. The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Once during week 1 and week 6 of treatment, samples of prepared formulations were analysed for verification of concentration.
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred.
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy. Dose volumes were adjusted once per week for each animal according to the last recorded
body weight.

Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for each animal according to the last recorded body weight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
The test item was administered orally by gavage to parental animals at 5 mL/kg body weight.
The dosages, selected in consultation with the Sponsor, were 100, 300 and 1000 mg/kg/day.

Examinations

Maternal examinations:
yes
Ovaries and uterine content:
yes
Fetal examinations:
yes
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test if n was more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The criteria for statistical significance were p<0.05 and p<0.01.
The mean value, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight gain were lower in the high dose group compared to controls throughout
the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was reduced in the high dose group compared to controls.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The main relevant change was an increased value of bile acids in treated groups compared to controls with clear dose-relation in males.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant higher kidneys weight was observed in high dose males and females compared to controls. In addition, thymus weight was significantly decreased in high dose males.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Stomach (non-glandular)
The treatment-related change seen in the high dosed animals (1/10 and 5/10, respectively in males and females), consisted of mild diffused hyperplasia of the squamous epithelium in the non-glandural stomach, which was associated with mild thickening (i.e., hyperkerathosis) of the keratin layer. This change was not associated with any indication of inflammation and/or ulceration.

Liver
In 3/10 high dose animals (females), minimal degree of multifocal perilobular hepatocytic vacuolaiton, which is suggested to be consistent with fatty change, was noted. The vacuoles were of mixed type (i.e., micro- and macrovesicular) and no presence of inflammation and/or necrosis was noted.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
The percentage of cumulative pup loss on Day 4 post partum starting from the total litter size at birth, was increased in the high dose group.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced litter and mean pup weights were found in the high dose group compared to controls.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Reduced litter and mean pup weights were found in the high dose group compared to controls.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The percentage of cumulative pup loss on Day 4 post partum starting from the total
litter size at birth, was increased in the high dose group.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Reduced litter and mean pup weights were found in the high dose group compared to controls.
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
On Day 4 post partum, one high dose female showed a very high percentage of cumulative pups loss (>50%). The mean value was indeed increased in the high dose group but no statistical significance was reached.
External malformations:
no effects observed
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
not specified

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
1,4-Butanediol dimethacrylate did not show developmental toxicity via oral gavage to rats at doses as high as 300 mg/kg bw/day in the described reproductive/developmental toxicity screening study according to OECD 422. At 1000 mg/kg/d, reduced litter and mean pup weights were observed.
The pronounced reduction of body weight gain of the premating animals indicates that the reduced reproduction success is likely a secondary effect of general systemic toxicity resulting in a poor condition of the parent females.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD Guideline 422 (22 March 1996) 1,4-Butanediol dimethacrylate was administered to 10 Hsd: Sprague Dawley SD rats/sex/dose orally by gavage at dose levels of 0 (control), 100, 300 and 1000 mg/kg bw/d. The treatment schedule included 2 weeks before pairing, during pairing, post coitum and post partum periods up to day 3 post partum. Animals were administered for approximately 5 and 8 weeks for males and females, respectively.


 


No mortality occurred in the study. A total of 8 females were found not pregnant at necropsy: one each in the control and low dose groups and 6 in the high dose group.
The number of females with live pups on Day 4 post-partum were: 9 in the control group, 9 in the low dose group, 10 in the mid-dose group and 4 in the high dose group.
No clinical signs of toxicological significance were reported.


Body weight and body weight gain were lower in the high dose group compared to controls throughout the study. 
Food consumption was reduced in the high dose group compared to controls. No relevant differences were noted in all parameters investigated between control and treated groups. In accordance with the expert statement, the reduced food consumption and the body weight loss is a direct consequence of the emerging Methacrylic acid (see Chapter 7.1.1. Toxicokinetics and expert statement, Chapter 13.2). 
The main relevant change was an increased value of bile acids in treated groups compared to controls with a clear dose-relation in males. 
No changes were recorded in urinalysis.
Measurements of oestrus cycle, pre-coital intervals and copulatory index did not show differences between treated and control groups. On the contrary, fertility index was markedly reduced in the high dose group (40% compared to 90% of the control group). In accordance with the expert statement, the reduced fertility index is the direct of consequence of maternal toxicity due to the markedly reduced body weight/ food consumption. The markedly reduced body weight leads to the interruption of the Leptin-Ghrelin-Kisspetin-GnRH hormon axis (see expert statement, Chapter 13.2). 


No significant differences were observed in the number of implantations, corpora lutea, total litter size, pre-implantation loss, pre-birth loss and gestation length between control and treated groups.
Reduced litter and mean pup weights were found in the high dose group compared to controls. The percentage of cumulative pup loss on Day 4 post partum starting from the total litter size at birth, was increased in the high dose group. The reduced litter and mean pup weight is also a secondary effect of the reduced maternal body weight and pup's survial is also directly linked with their general condition after birth. No differences were found in sex ratio between the groups.
Small pups were generally observed in all groups including the control group. Cold to touch and apparently no food intake were the signs noted in pups of the treated groups only.
No relevant differences were recorded in decedent pups between the groups.
No abnormalities were observed in pups sacrificed at term.
Terminal body weight was lower in the high dose group compared to controls and this difference was statistically significant in females. The significantly reduced body weight in females leads to the interruption of the Leptin-Ghrelin-Kisspetin-GnRH hormon axis. This is well-known and widely described in the literature.  
Statistically significant higher kidneys weight was observed in high dose males and females compared to controls. In addition, thymus weight was significantly decreased in high dose males. The higher kidney weights are considered to the consequence of the high volume intake due to the gavage administration (10 ml/kg bw) and not adverse since they are not accompanied by microscopic changes. The reduced thymus weight (thymus atrophy) is considered to be stress-related due to the hyperplasia in the forestomach and consequently reduced food consumption and body weight (see expert statement, Chapter 13.2).
No treatment-related changes were noted at macroscopic examination.
Treatment-related findings at microscopic observations were limited to the high dosed animals and were seen in the stomach of both sexes and in the liver of the females only. The treatment-related change seen in the high dosed animals (1/10 and 5/10, respectively in males and females), consisted of focal hyperplasia of the squamous epithelium in the non-glandular stomach, which was associated with thickening (i.e., hyperkeratosis) of the keratin layer. This change was not associated with any indication of inflammation and/or ulceration.


The findings in the forestomach of the animals are considered as main result of the study, treatment-related and adverse. The reason for the adverse alteration in the forestomach is the emerging Methacrylic acid due to the well-known, well-described and widely accepted metabolism of Methacrylates. (see Chapter 7.1.1., Toxicokinetics, see expert statement, Chapter 13.2, see CLH proposal of Finnish MSCA, Chapter 13.2).


In 3/10 high dose animals (females), minimal degree of multifocal perilobular hepatocytic vacuolation, which is suggested to be consistent with fatty change, was noted. The vacuoles were of mixed type (i.e., micro- and macrovesicular) and no presence of inflammation and/or necrosis was noted. The minimal degree of multifocal perilobular hepatocytic vacuolation in the liver of females which is not accompanied with necrosis or functional restrictions is considered to be not adverse since it is part of the normal background changes in the liver observed in animal studies (see expert statement, Chapter 13.2). 


Evaluation of the spermatogenic cycle did not show differences between the groups. Regular layering in the germinal epithelium was noted.


 


Conclusions


On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity could be considered 300 mg/kg bw/d for both sexes. The NOAEL on reproduction/developmental toxicity could be considered 1000 mg/kg/day.


 


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