Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

1,4-BDDMA was studied in an OECD 422 combined repeat dose and reproductive/developmental toxicity screening test (RTC, 2013). Groups of 10 male and 10 female rats were administered by gavage at dose levels of 0, 100, 300, or 1000 mg/kg/day. Male rats were dosed for 33 days and female rats were dosed from 14 days prior to mating through Day 3 of lactation. On the basis of the results obtained in this study, there were significant signs of toxicity in the 1000 mg/kg/day group for males and females with histopathological findings in the liver (females) and stomach (males and females). A reduction of the fertility index has been observed at 1000 mg/kg/day. The NOAEL for effects on fertility as well as for parental toxicity was found to be 300 mg/kg/day.

1,4 -Butanediol dimethacrylate will rapidly be hydrolyzed by unspecific carboxyl esterases in the liver into methacrylic acid and 1,4 -butanediol (see chapter Toxikokinetics and the Category document, respectively).

Therefore corresponding to the requirements of Annex X higher studies were covered by a read across approach with the hydrolyses products methyl methacrylate (the metabolite donor substance for methacrylic acid) and 1,4-Butanediol respectively the analogous metabolite 1,3-Butanediol.

Methyl methacrylate

No reproductive effects were observed in a 2-generation oral gavage study with rats acc. OECD 416 (BASF 2009) up to 400 mg/kg bw/day with methyl methacrylate. NOAEL for fertility: 400 mg/kg bw/day.

1,4-Butanediol

No reproductive effects were observed in a repeated dose reproduction/developmental toxicity screening test acc. OECD 422 in rats (MHW 1999) with the alcohol 1,4 Butanediol. NOAEL ferility: 800 mg/kg bw/day.

1,3 -Butanediol

The effect of 1,3-BD on reproductive performance as well as its teratogenic, dominant lethal and cytogenetic effects were studied in a five generation of Wistar rats (Hess et al. 1981). The study is rated with a reliability of 2 (publication with some limitations in documentation and result evaluation).Animals of both sexes were fed either control diet or diet supplemented with 1,3-BD at dose levels of 5, 10 or 24% of the diet by weight. Reproduction and lactation parameters were comparative to controls for four of five generations of dams and pups. In contrast, the pregnancy rate of FlA rats decreased during five successive mating cycles; no pups were obtained in the high-dose level group of the fifth series of litters (F2E generation). Excluding this group, the viability of F2 generation pups revealed no significant differences between litters or between control and test groups. A NOAEL for parenteral animals of 10% based on the reduced pregnancy rate was identified in this study. The NOAEL for the offspring is considered to be 24% in the diet.

In summary based on available data on 1,4 -Butanediol dimethacrylate and its metabolite1,4 -Butanediol and the metabolite donor substance Methyl methacrylate or the read across with the related alcohol 1,3-Butanediol, 1,4 -Butanediol dimethacrylate did not show reproductive effects.

Compliance to REACh requirements

The screening study requirement is covered with a reliable OECD 422 oral rat study, performed with the substance itself. The reproduction toxicity requirements are covered with a reliable two generation study in rats with the methacrylic metabolite donor MMA and a reliable five generation study with rats with the alcohol metabolite analogue 1,3-BD. The read across is done with a high level of confidence (see chapter Toxikokinetics and the Category document, respectively).

According to column 1 of Annex IX, section 8.7.3 of REACH regulation, a decision on the need to perform a second two generation study at this tonnage level should be based on the outcome of all other relevant available data. In the available data set on 1,4-Butanediol dimethacrylate itself, its metabolites/ metabolite donor substances and the analogous metabolites mentioned above, 1,4 -Butanediol dimethacrylate did not show reproductive effects in rats. The available data are sufficient for evaluation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 20 April to 25 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy. Animals were bred by Harlan Laboratories Netherland - Kreuzelweg 53, 5961 NM Horst
P.O. Box 6174 NL 5960 AD Horst (Netherlands).
- Age at study initiation: 9 to 10 weeks old (day of allocation)
- Weight at study initiation: (P) Males: 279-280g; Females: 214-216g (day of allocation)
- Fasting period before study: no
- Housing: 5 sex/cage
- Diet (e.g. ad libitum): ad libitum except for clinical pathology
- Water (e.g. ad libitum): ad libitum except for clinical pathology
- Acclimation period: circa 3 weeks

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

IN-LIFE DATES: from 26 April (day of allocation) to 25 June 2012
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.
Details on mating procedure:
- M/F ratio per cage: one male to one female
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: single
- Any other deviations from standard protocol:no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (check of concentration and homogeneity).
The stability was found to be 24 hours at room temperature in the concentration range of 20 to 200 mg/mL.
Samples of the formulations prepared on Weeks 1 and 6 (when all females were present) were also analysed to check the concentration and homogeneity.
The overall results of the analyses were within the limits of acceptance stated in RTC’s SOPs for concentration (90-110%) and homogeneity (CV <10%).
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. 90770), in the range from 1.0 to 200 mg/mL. The software used for this activity was the Empower® Pro build No. 2154.
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for 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 2 consecutive weeks prior to pairing and thereafter during pairing (up to Day 40 of pairing for female no. 90760079 which did not mate), post coitum (up to Day 26 for non pregnant females) and post partum periods until Day 3 post partum. 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.
Frequency of treatment:
Once a day
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:
3 groups of 10 males and 10 females each. A similar constituted control group (Group 1) received the vehicle alone.
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.

Parental animals: Observations and examinations:
Mortality

Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Clinical signs

All clinical signs were recorded for individual animals.
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were record.
Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

Clinical observations (Functional Observation Battery Tests)

Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
All observations were recorded for individual animals.

Grip strength and sensory reactivity to stimuli

Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order. For males the tests were performed 4 days before necropsy and for females on Day 3 post partum.

Motor activity assessment (MA)

Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. For males the tests were performed 4 days before necropsy and for females on Day 3 post partum.

Food consumption

The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period following allocation. The interval between allocation and treatment initiation was less than a full week. Individual food consumption for the females was measured on Days 7, 1 4 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

Body weight

Males were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter and at termination.

Females were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter until pairing and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.
Body weight measured weekly during the mating phase is not presented in this report but retained and archived as study raw data.
As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (all females with viable litters with the exception of one not pregnant female of Group 4) randomly selected from each group, under condition of food deprivation.
The blood samples collected were divided into tubes as follows:

EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests


The measurements performed on blood samples are listed below:

Haematology

Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count - Neutrophils
- Lymphocytes
- Eosinophils
- Basophils
- Monocytes
- Large unstained cells
Platelets

Coagulation tests

Prothrombin time

Clinical chemistry

Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

Urinalysis (Only males)

At the same time interval as the clinical pathology investigations, individual overnight urine samples were also collected from the same animals under the same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.

Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood

The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:

Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components

Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made.
The vaginal smear data were examined to determine the following:

a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations:
testis weight, epididymis weight, morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle)
Litter observations:
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1 and 4 post partum.
Pups dying during the lactation period were weighed before the despatch to necropsy (these data are not tabulated in this report but archived together with all raw data).
Observation was performed once daily for all litters.
Postmortem examinations (parental animals):
Parental animals were killed by exsanguination under isofluorane anaesthesia.
Pups were euthanised by intraperitoneal injection of Thiopental.

Parental males:
The males were killed after the mating of all females (after 33 days of treatment).

Parental females:
The females with live pups were killed on Day 4 post partum.
One Group 4 female (animal no. 90760079) showing no evidence of copulation was killed after 26 days post coitum from the last day of the mating session.
The females which did not give birth 25 days after positive identification of mating were killed shortly after.

The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Females:
All females were examined also for the following:

a) external and internal abnormalities;
b) number of visible implantation sites (pregnant animals);
c) number of corpora lutea (pregnant animals).

Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.
Organ weights

From all animals completing the scheduled test period, the organs were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved

Samples of all the tissues listed in section 4.5.6 were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).

Histopathological examination

The tissues required for histopathological examination are listed in section 4.5.6. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

In the first instance the examination was restricted as detailed below:

a) Tissues from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group.
b) All abnormalities in all groups.

The examination of stomach (both sexes) and liver (only females) was extended to include the remaining 5 males and 5 females (animals not evaluated for clinical pathology) of the control and high dose group.
In addition, the examination of stomach (both sexes) and liver (only females) was extended to animals of the other dose groups since possible treatment-related changes were observed in the high dose group.

Postmortem examinations (offspring):
All pups found dead in the cage were examined for external and internal abnormalities. Sex confirmation by gonadal inspection was also performed.
All live pups sacrificed at termination were examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was 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 non-parametric Kolmogorov-Smirnov test (for stomach and liver).
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 non-parametric version of the Williams test.
The criterion for statistical significance was p<0.05 or p<0.01.
Reproductive indices:
Males
Copulatory Index (%)=no. of animals mated/no. of animals paired x 100
Fertility Index (%)=no. of males which induced pregnancy/no. of males paired x 100

Females
Copulatory Index (%)=no. of animals mated/no. of animals paired x 100
Fertility Index (%)=no. of pregnant females/no. of females paired x 100

Males and females
Pre-coital Interval=Mean number of days between pairing and mating
Offspring viability indices:
Pre-implantation loss was calculated as a percentage from the formula:

(No. of corpora lutea - no. of implantations)/No. of corpora lutea x 100

Pre-birth loss was calculated as a percentage from the formula:

(No. of visible implantations - total litter size at birth)/No. of visible implantations x 100

Pup loss at birth was calculated as a percentage from the formula:

(Total litter size - live litter size)/Total litter size x 100

Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:

(Total litter size at birth - live litter size at Day 4)/Total litter size at birth x 100

Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.
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
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
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
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).

No significant differences were observed in the number of implantation, corpora lutea, total litter size, pre-implantation loss, pre-birth loss and gestation length between control and treated groups.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
reproductive performance
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Cold to touch and apparently no food intake were the signs noted in pups of the treated groups only.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Body weight and 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.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
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. No differences were found in sex ratio between the groups.
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
Other effects:
no effects observed
Description (incidence and severity):
No significant differences were observed in the number of implantation, corpora lutea, total litter size, pre-implantation loss, pre-birth loss and gestation length between control and treated groups.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: Reduced litter and mean pup weights in high dose group. Percentage of cumulative pup loss on Day 4 post partum starting from the total litter size at birth, increased in high dose group.
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

 Oestrus cycle – Pre-pairing period - Group summary data

 

 STUDY NO.: 90760

 -----------------------------------------------------------------------------------------------------------------------------------

           Group 1              Group 2             Group 3             Group 4

 Animal   Oestrus    Animal   Oestrus   Animal   Oestrus   Animal   Oestrus

 Number   Cycles     Number   Cycles    Number   Cycles    Number   Cycles

 -----------------------------------------------------------------------------------------------------------------------------------

 90760001    2      90760021     3      90760041    3      90760061    2

 90760003    3      90760023     3      90760043    3      90760063    1

 90760005    4      90760025     3      90760045    4      90760065    3

 90760007    2      90760027     2      90760047    3      90760067    2

 90760009    3      90760029     4      90760049    2      90760069    3

 90760011    2      90760031     2      90760051    1      90760071    3

 90760013    2      90760033     1      90760053    1      90760073    1

 90760015    0      90760035     2      90760055    2      90760075    4

 90760017    4      90760037     2      90760057    3      90760077    2

 90760019    2      90760039     3      90760059    3      90760079    3

 

  Mean     2.4                  2.5                 2.5                 2.4

-----------------------------------------------------------------------------------------------------------------------------------

 Note: The number of oestrus cycles is based on the number of non sequential days the dams were in oestrus.

Reproductive performance

Reproductive parameters of males - Summary

 

-----------------------------------------------------------------------------------------------------------------------------------

                              Group              1          2         3          4

-----------------------------------------------------------------------------------------------------------------------------------

 

   Copulatory Index%                    100.0     100.0      100.0      90.0

-----------------------------------------------------------------------------------------------------------------------------------

 

   Fertility Index%                       90.0      90.0      100.0      40.0

-----------------------------------------------------------------------------------------------------------------------------------

 

Reproductive parameters of females - Summary

-----------------------------------------------------------------------------------------------------------------------------------

                              Group              1          2         3          4

-----------------------------------------------------------------------------------------------------------------------------------

 

   Copulatory Index%                    100.0     100.0      100.0      90.0

-----------------------------------------------------------------------------------------------------------------------------------

 

   Fertility Index%                           90.0      90.0      100.0      40.0

-----------------------------------------------------------------------------------------------------------------------------------

  

Conclusions:
On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity could be considered 300 mg/kg/day.
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.
No changes of toxicological significance were found.
The main relevant change was an increased value of bile acids in treated groups compared tocontrols 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).
No significant differences were observed in the number of implantation, 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.
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.
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.
No treatment-related changes were noted at macroscopic examination.
Treatment-related findings at microscpopic 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.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/orulceration.
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/ornecrosis was noted.
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 and reproduction/developmental toxicity could be considered 300 mg/kg/day for males and females. 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.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Although the study was only reported in 2009 it was already commisioned before the establishment of ECHA and therefore no test proposal could be submitted.
Justification for type of information:
Read across from the methacrylic metabolite donor substance.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1ff

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, table in chapter 1.2 and endpoint specific chapters
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes (incl. QA statement)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 37 (±1) days at the beginning of treatment; (F1) x wks
- Weight at study initiation: (P) Males: 127.5 - 151.0 g; Females: 110.5 - 145.1 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages (Becker & Co., Castrop-Rauxel, Germany), floor area of about 800 cm², with the following exceptions: 1) During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. 2) Pregnant animals and their litters were housed together until PND 21 (end of lactation).
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water: ad libitum
- Acclimation period: (P) about 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10 or 15 times
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance suspensions were prepared at the beginning of the administration period and thereafter at intervals that took into account the analytical results of the stability verification. For the test substance preparation, the specified amount of test substance was weighed into an Erlenmeyer flask, topped up (shortly under the marking) with 1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The Erlenmeyer flask was sealed and the preparation was intensely mixed with a magnetic stirrer. A magnetic stirrer was used to keep the preparations homogeneous during treatment of the animals.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (= GD 0)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance preparations were sent to the analytical laboratory ten times during the study period (among other things at the beginning and towards the end) for verification of the concentrations. The samples, which were taken for the concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (50 and 400 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.
Duration of treatment / exposure:
until one day before sacrifice
Frequency of treatment:
once daily
Details on study schedule:
- F1 parental animals not mated until 75 days after selected from the F1 litters.
- Selection of parents from F1 generation after weaning (PND 21).
- Age at mating of the mated animals in the study: [...] weeks
Remarks:
Doses / Concentrations:
0, 50, 150, 400 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
F0 generation parental animals: 25
F1 generation parental aniumals: 25
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days or once daily (Saturday, Sunday or on public holidays)


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: first day of the premating period and then once a week at the same time of the day (in the morning) until sacrifice. The following exceptions are notable for the female parental animals: 1) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. 2) Females showing no positive evidence of sperm in vaginal smears were weighed once a week during the mating interval (solely for calculation of dose volume). 3) Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 14 and 21. 4) Females without litter were weighed once a week during the lactation phase (solely for calculation of dose volume).



OTHER:
- Food consumption: In general, food consumption was determined once a week for the male and female F0 and F1 parental animals. After the 10th test week, food consumption of the females during pregnancy (animals with evidence of sperm) was determined weekly for GD 0-7, 7-14 and 14-20. During the lactation period (animals with litter) food consumption was determined for PND 1-4, 4-7, 7-14 and 14-21.
Oestrous cyclicity (parental animals):
Estrous cycle length and normality were evaluated daily for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating. The evaluations were continued throughout the mating period until the female exhibited evidence of mating. Moreover, at the scheduled necropsy a vaginal smear was microscopically examined to determine the stage of the estrous cycle for each F0 and F1 female.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight, cauda epididymis weight, prostate weight, seminal vesicles including coagulation glands weight, sperm head count in testis, sperm head count in cauda epididymis, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, clinical symptoms, sexual maturation


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE AND GROSS NECROPSY
All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined: Anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, cauda epididymis, prostate, seminal vesicles including coagulation glands, ovaries, uterus, spleen, brain, pituitary gland, thyroid glands (with parathyroid glands). The following organs or tissues of the F0 and F1 generation parental animals were fixed in 4% neutral buffered formaldehyde solution or in BOUIN’s solution, respectively: Vagina, cervix uteri, uterus, ovaries (BOUIN), oviducts, left testis (BOUIN), left epididymis (BOUIN), seminal vesicles, coagulation glands, prostate, pituitary gland, adrenal glands, liver, kidneys, spleen, brain, thyroid glands (with parathyroid glands), all gross lesions. After fixation, the organs fixed in BOUIN´s solution were embedded in Paraplast. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.

OTHER:
- Differential Ovarian Follicle Count (DOFC) in F1 generation: From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, respectively, at least 100 µm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated. Primordial follicles and growing follicles were counted by light microscope (magnification: 100x) on each of these slides, – according to the definitions given by Plowchalk et al. (PLOWCHALK, D. R., B. J. SMITH, and D. R. MATTISON: Assessment of Toxicity to the Ovary Using Follicle Quantitation and Morphometrics. In: Methods in Toxicology, Vol. 3, Part B: Female Reproductive Toxicology (J. J. HEINDEL and R. E. CHAPIN, Editors), p. 57-68, 1993, Academic Press). To prevent multiple counting on serial slides – especially of the growing follicles – only follicles with an oocyte with visible chromatin on the slide were counted. The number of each type of follicle was recorded individually for ovary 1 and ovary 2 of every animal on any of the slide levels (level 1-10), giving in summary the incidence of each type of the follicles by using EXCEL sheets for the reporting of the results. Finally, the results of all types of follicles were summarized for all animals per group in dose groups 10 and 13. As primordial follicles continuously develop into growing follicles, the assessment of the follicles was extended to the combined incidence of primordial plus growing follicles. In general, the fifth slide of the left and right ovary was evaluated for histological findings. Whenever the diagnosis: ”no abnormalities detected” was used for the ovaries, this implicates all functional statuses of follicles, especially corpora lutea, were present. An attempt was made to correlate gross lesions with histopathological findings.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age. All F2 offspring were sacrificed after weaning (~ PND 21).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- All pups were examined externally and eviscerated; their organs were assessed macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
Animals with notable findings or abnormalities were further evaluated on a case-by-case basis, depending on the type of finding noted. After the scheduled sacrifice the brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. Normally, the first male and the first female pups/litter were taken for these determinations. For the calculation of the relative organ weights, the pup body weights, determined routinely during the in-life phase on PND 21, were used.
Reproductive indices:
- Male reproduction data: The mating partners, the number of mating days until vaginal sperm could be detected in the female, and the gestational status of the female were noted for F0 and F1 breeding pairs. For the males, mating and fertility indices were calculated for F1 and F2 litters according to the following formulas: Male mating index (%) = (number of males with confirmed mating)/(number of males placed with females) x 100. Male fertility index (%) = (number of males proving their fertility)/(number of males placed with females) x 100.
- Female reproduction and delivery data: The mating partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 and F1 females. For the females, mating, fertility and gestation indices were calculated for F1 and F2 litters according to the following formulas:
Female mating index (%) = (number of females mated)/(number of females placed with males) x 100.
Female fertility index (%) = (number of females pregnant)/(number of females mated) x 100.
Gestation index (%) = (number of females with live pups on the day of birth)/(number of females pregnant) x 100.
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 and F2 litters according to the following formula:
Live birth index (%) = (number of liveborn pups at birth)/(total number of pups born) x 100.
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = (number of implantations – number of pups delivered)/(number of implantations) x 100.
Offspring viability indices:
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21. Furthermore, viability and lactation indices were calculated according to the following formulas:
Viability index (%) = (number of live pups on day 4 (before culling) after birth)/(number of live pups on the day of birth) x 100.
Lactation index (%) = (number of live pups on day 21 after birth)/(number of live pups on day 4 (after culling) after birth) x 100.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test group 03 (400 mg/kg bw/d)
• Statistically significantly decreased food consumption in parental males during premating weeks 5 - 10 (up to 7%)
• Statistically significantly decreased food consumption in parental females during premating weeks 1 - 3 (up to 6%), weeks 5 - 8 (up to 7%) and weeks 9 - 10 (about 6%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 7 (about 10%)
• Statistically significantly decreased food consumption in parental females during lactation days 4 - 7 (about 7%)

Test group 02 (150 mg/kg bw/d)
• Statistically significantly decreased food consumption in parental females during premating weeks 1 - 2 (about 5%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 7 (about 7%)

Test group 01 (50 mg/kg bw/d)
• no test substance-related adverse effects/findings
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Under the conditions of the present 2-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 400 mg/kg bw/d for the F0 parental rats, the highest dose tested.
The NOEL (no observed effect level) is 50 mg/kg bw/d for the F0 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.
The NOAEL for fertility and reproductive performance for the F0 parental rats is 400 mg/kg bw/d, the highest dose tested.
Dose descriptor:
NOEL
Remarks:
food consumption
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: adverse effects on food consumption observed at the LOEL of 150 mg/kg bw/day in the F0 parental females
Remarks on result:
other: Generation: P and F1 parental animals (migrated information)
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Generation: P and F1 parental animals (migrated information)
Dose descriptor:
NOAEL
Remarks:
General systemic toxicity
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed
Remarks on result:
other:
Remarks:
NOAEL (no observed adverse effect level) for general, systemic toxicity is 400 mg/kg bw/d for the F0 and F1 parental rats, the highest dose tested.
Dose descriptor:
LOEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
food consumption and compound intake
Remarks on result:
other:
Remarks:
consequence of reduced appetite observed in the F0 parental females
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test group 03 (400 mg/kg bw/d)
Statistically significantly decreased food consumption in parental males during premating weeks 0 - 1 (about 14%) and weeks 8 - 10 (up to 7%)
• Statistically significantly decreased food consumption in parental females during premating weeks 0 - 1 (about 10%) and weeks 9 - 10 (about 8%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 14 (up to 8%)
• Statistically significantly decreased body weights in parental males during weeks 0 - 5 (up to 17%) and weeks 10 - 11 (up to 6%)
• Statistically significantly decreased body weights in parental females during premating weeks 0 - 1 (up to 16%)

Test group 02 (150 mg/kg bw/d)
• no test substance-related adverse effects/findings

Test group 01 (50 mg/kg bw/d)
• no test substance-related adverse effects/findings
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
The NOEL (no observed effect level) is 50 mg/kg bw/d for the F1 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.
The NOAEL for fertility and reproductive performance for the F1 parental rats is 400 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental toxicity, in the F1 of the test substance is 400 mg/kg bw/d, the highest dose tested.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
food consumption and compound intake
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Remarks on result:
other:
Remarks:
highest dose tested
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmenatl toxicity
Reproductive effects observed:
no

- Analytics:

The various analyses:

  • demonstrated the stability of the test substance preparations over a period of 7 days at room temperature
  • confirmed the homogeneous distribution of the test substance in the vehicle (1% Carboxymethylcellulose suspension in drinking water and a few drops Cremophor EL and one drop hydrochloric acid)
  • showed that the prepared concentrations were close to the expected values ranging between 86.8 and 113.2% of the nominal concentrations

Analytical values (range):

Test group

Nominal Dose
(mg/kg bw/d)

Analytical Dose
(mg/kg bw/d)
[minimum]

Analytical Dose (mg/kg bw/d)
[maximum]

% Nominal Dose
[minimum]

% Nominal Dose
[maximum]

00 / 10

0

0

0

 

   

01 / 11

50

43.40

46.61

86.8

93.2

02 / 12

150

132.90

169.80

88.6

113.2

03 / 13

400

359.20

379.90

89.8

95.0

 

Conclusions:
The NOAEL for general, systemic toxicity is 400 mg/kg/d for the parental rats in the highest dose tested.
The NOEL 50 mg/kg bw/day for the P parental rats, based on effects on food consumption observed at the LOAEL of 150 mg/kg bw/day in the P parental females.
The NOAEL for fertility and reproductive performance for the P and F1 parental rats is 400 mg/kg bw/day, the highest dose tested.
The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance is 400 mg/kg bw/day, the highest dose tested.
Executive summary:

The study was performed according to OECD TG 416 in compliance with GLP. Methyl Methacrylate was administered to groups of 25 male and 25 female healthy young Wistar rats (P parental generation) as an aqueous preparation by stomach tube at dosages of 0; 50; 150 and 400 mg/kg body weight/day. At least 73 days after the beginning of treatment, P animals were mated to produce a litter (F1). Mating pairs were from the same dose group and F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 25 males and 25 females, selected from F1 pups to become F1 parental generation, were treated with the test substance at dosages of 0; 50; 150 and 400 mg/kg body weight/day post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 adult animals.

Control parental animals were dosed daily with the vehicle (1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop hydrochloric acid).

The mid- and high-dose parental animals (400 mg/kg bw/d) showed clinical signs of systemic toxicity. The only relevant clinical observation was temporary salivation during a short period after dosing, which is considered to be test substance-induced. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding.

In the mid- and high-dose (150 and 400 mg/kg bw/d) P generation animals, dose-related intermittent reductions of food consumption were noted, either during premating, gestation and lactation phases of this study. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group.

High dose F1 parental males had statistically significant lower body weights during several study segments, which led to a statistically significant reduction of the mean terminal body weight resulting in secondary weight changes of brain.

High dose parental females had statistically significant lower body weights during the first weeks after weaning. This weight decrease during major phases of sexual maturation led to an apparent marginal delay of vaginal patency. This minor delay did, however, not result in any corroborative pathological findings nor did it adversly effect F1 female cyclicity, fertility and reproduction. Thus, an influence of the test substance on female sexual maturation is not assumed.

Pathological examinations revealed no test-substance-related changes in organ weights, gross lesions, changes in differential ovarian follicle counts or microscopic findings, apart from an increase in kidney and liver weights in male and female animals in both generations which is presumably related to the treatment. There was no histopathologic lesion observed, that could explain the weight increase. It is regarded to be an adaptive change, most likely caused by an increase in metabolic activity in the two organs, which does not lead to histopathologic findings. It is not regarded to be an adverse effect.

There were no indications from clinical examinations as well as gross and histopathology, that the administration of methyl methacrylate via the diet adversely affected the fertility or reproductive performance of the P or F1 parental animals up to and including a dose of 400 mg/kg bw/day. Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the rats of all test groups and ranged within the historical control data of the test facility.

All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offspring up to a dose level of 400 mg/kg bw/day. Up to this dose level, the test substance did not adversely influence pup viability and pup body weights. Sex ratio and sexual maturation was not directly affected at any dose level, inclusive the high-dose group (400 mg/kg bw/day).

Conclusion:

Under the conditions of the present 2-generation reproduction toxicity study the NOAEL(no observed adverse effect level) forgeneral, systemic toxicityis 400 mg/kg bw/d for the parental rats, the highest dose tested.

The NOEL (no observed effect level) is 50 mg/kg bw/d for the F1 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.

The NOAEL for fertility and reproductive performance for the F1 parental rats is 400 mg/kg bw/d, the highest dose tested.

 The NOAEL for developmental toxicity, in the F1 of the test substance is 400 mg/kg bw/d, the highest dose tested.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, only original in Japanese and abstracts in English available
Justification for type of information:
Read across from the alcohol metabolite.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1ff

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, table in chapter 1.2 and endpoint specific chapters
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan (Ltd.) Atsugi breeding center
- Age at study initiation: 7 weeks
- Weight at study initiation: male: 297-305 g; female: 256-277 g
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ±
- Humidity (%): 50-60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Amount of vehicle (if gavage): 5 ml
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 15 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Males: 42 days
Females: from 14 days prior to mating to day 3 of lactation
Frequency of treatment:
daily
Details on study schedule:
Exposure period:
Males: For 2 weeks prior to mating and 2 weeks of mating
Females: For 2 weeks prior to mating and 2 weeks of mating and throughout pregnancy antil day 3 postpartum
Remarks:
Doses / Concentrations:
200, 400, 800 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
13 males, 13 females
Control animals:
yes, concurrent vehicle
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes /
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): YES
P males during treatment period, P females during pre-mating period, pregnant period and lactation period
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Postmortem examinations (parental animals):
bladder, liver, kidney, heart, spleen, thymus, testes, epididymides and Haderian gland were observed
Postmortem examinations (offspring):
viablity, body weight on day 4, embodyment
Reproductive indices:
- number of mated pairs
-number of copulated pairs
-copulation index
-number og pregnat animals
-fertility index
-pairing days until copulation
- times of vaginal estrus
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were suppressed at 400 and 800 mg/kg during the early period of administration. Food consumption also decreased according to body weight gain
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were suppressed at 400 and 800 mg/kg during the early period of administration. Food consumption also decreased according to body weight gain
Food efficiency:
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Transient hyperactivity only just after administration was observed at 200 mg/kg/day. At 400 mg/kg, activities were rather suppressed than increased although hyperactivity was also observed after a few doses. At 800 mg/kg, toxic signs observed were more severe and some animals were even comatose after showing hypoactivity and recumbency. By 5 hours after dosing these signs disappeared and animals recovered to normal.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the histopathological examination, diffuse transitional epithelial hyperplasia and fibrosis in the lamina propria of the urinary bladder were observed in the 400 and 800 mg/kg groups.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
The parental animals exhibited no alteration in reproductive parameters including the copulation index, ferility index, gestation length, numbers of corpora lutea or implantation, gestation lenghth, numbers of corpora lutea or implantation index, gestation index, delivery index and behavior at delivery and lactation.
Dose descriptor:
LOEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
neuropathology
other: Hyperactivity/ transient neurotoxic signs in central nervous system. No alteration in reproductive parameters.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other:
Remarks:
This change was considered to be secondary to maternal toxicity /reduced food consumption and body weight gain).
Reproductive effects observed:
no
Lowest effective dose / conc.:
800 mg/kg bw/day
Relevant for humans:
not specified

For reproductive toxicity, in the observation items related to fertility of the parent generation, change to related test substance administration was observed. The pup weight of the offspring after 4 days of lactation was reduced in the 800 mg / kg dose group in m,ales and females. Although this decline might be the average body weight of nursing four days of the mother animal is due to the fact was a low value, toxicological significance is unknown.

Executive summary:

An oral subacute toxicity study was performed in SD (Crj: CD) rats by an OECD 422 combined repeat dose and reproductive/developmental toxicity screening test. Administration was conducted at doses of 200, 400 or 800 mg/kg/day by gavage for 45 days in males and from 14 days before mating to day 3 of lactation in females. (MHW, Japan: 1999).

The parental animals exhibited no alteration in reproductive parameters including the copulation index, fertility index, gestation length, numbers of corpora lutea or implantation, implantation index, gestation index, delivery index, and behavior at delivery and lactation. Although neither the pup viability nor the incidence of morphological abnormalities was changed by administration of the compound, pup body weight was slightly but significantly decreased in the 800 mg/kg group. This change was considered to be secondary to maternal toxicity (reduced food consumption and body weight gain). LOAEL parental 200 mg/kg/day (male, female) due to neurotoxic effects (based on OECD SIDS review). NOAEL F1: 800 mg/kg.

Endpoint:
multi-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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
Remarks:
limitations: exposure duration not clearly stated, no statistical evaluation
Justification for type of information:
Read across from an analogous metabolite.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1ff

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, table in chapter 1.2 and endpoint specific chapters
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Five generation study with embedded continuous breeding study
GLP compliance:
not specified
Limit test:
no
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.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 7 d
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged:individually
Analytical verification of doses or concentrations:
not specified
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. After 11 weeks of feeding, 25 males and 25 females from each dosage group of F1A animals were randomly selected and paired to produce the F2 generation (no further information).
Frequency of treatment:
daily
Details on study schedule:
At 1-2 weeks after weaning of the first litters (F1A), each female of the F0 generation was mated with a different male and a second series of litters was produced (F1B). All animals of the F1B generation were discarded at weaning except for ten males per group, which were reared to sexual maturity and used in a dominant lethal test. Pubs of the F1A were reared to maturity. After 11 weeks of feeding, 25 males and 25 females from each dosage group of F1A animals were randomly selected and paired to produce the F2 generation. Five successive mating cycles were achieved with the F1A rats within a period of 77 weeks (F2A, F2B, F2C, F2D, F2E). The F2B, F2C, F2D and F2E were examined and sacrificed as part of the continuous breeding phase of the study, while the F2A litter was mated to produce the F3A and F3B litters. The F3A litter was used for the cytogenetic portion of the study and was mated to produce the F4A and F4B litter, which are indicated by the chart in the orginal paper to be part of the cytogenicity study.The pregnant dams of the F2A litters (producing the F3B) were divided in two groups: 1/4 were allowed to give birth normally and 3/4 were used for teratological examination on day 19 of gestation.
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:
25 rats per sex per dose groupe in the F0, F1A, F1B, F2A, F3A
Control animals:
yes, plain diet
Details on study design:
Previous chronic studies have been conducted by feeding 1,3-butanediol at levels up to 10% of the diet for rats and 3% for dogs, without obvious deleterious test effects. The present study was conducted to evaluate the effects of 1,3-butanediol on the reproductive performance of rats through five generations fed levels up to 24% of the diet by weight.
Positive control:
no
Parental animals: Observations and examinations:
After 4 weeks of feeding of the F0 the respective diets, blood samples were collected from ten rats per sex per group for determination of alkaline phosphatase, glucose, hematocrit, hemoglobin and total and differential leucocyte counts. Urine analysis of the same animals provided measurements of albumin, glucose, ketones, occult blood, pH, specific gravity and microscopic examination. For F1A rats which survived at least 66 weeks, the gonads and pituitary glands were examined microscopically. During the eleventh week of feeding of F1A animals blood and urine samples were collected from ten rats per sex per group and evaluated as mentioned above.
Litter observations:
viability, mean pub weight at day 4 and 21 post partum
Postmortem examinations (parental animals):
histopathologic examination of the testes or ovaries and pituitary glands of the F1A
Reproductive indices:
fertility (percent matings resulting in pregnancies) and gestation indices (percent pregnancies resulting in litters cast alive)
Offspring viability indices:
- percent pubs cast alive that survived to 4 days
- percent pups alive at 4 days that survived to 21 days
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced weight gain in males of the highest dose group of the F1A, F1B, F2A and F3A generation (except F0).
Food efficiency:
no effects observed
Description (incidence and severity):
The efficiency of food utilization through 10 weeks of post-weaning remained constant for all generations of both sexes and was not affected by the level of 1,3-butanediol in the diet.
Haematological findings:
no effects observed
Description (incidence and severity):
Hematology showed no trends associated with treatment for the F0, F1, F2 and F3 generation animals.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Blood chemistry showed no trends associated with treatment for the F0, F1, F2 and F3 generation animals.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalyis showed no trends associated with treatment for the F0, F1, F2 and F3 generation animals.
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
During five successive mating cycles of F1A rats, a gradual decrease in the pregnancy rate was seen. Both the number of pregnant females and the fertility index appeared to be dose-related for several series of F2 litters, especially F2D and F2E. For the fifth series of litters, no pups were obtained in the highest-dose group. However, the gestation, viability and lactation indexes, as well as the mean pup body weights at 4 and 21 days showed no significant differences between specific litter series or between control and test groups (excluding high-dose animals of the fifth series of litters). No significant treatment-related differences were noted on histopathologic examination of testes or ovaries and pituitary glands as a possible explanation of the observed reproductive failure during the fifth cycle.For the other three generations of dams and pups, no significant dose-related trends were observed for the reproduction and lactation parameters, as described above.
Dose descriptor:
NOAEL
Effect level:
10 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
reproductive performance
other: NOAEL for all parental generations
Critical effects observed:
no
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains of male rats in all four F generations were slightly depressed, with an apparent dose relationship. (see P0)
no treatment related effects in offspring
Dose descriptor:
NOAEL
Generation:
other: F2A-F2E, F3A-F3B, F4A-F4B
Effect level:
24 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
no
Reproductive effects observed:
no
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD Guideline studies, GLP and peer reviewed studies according NTP protocol.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Developmental toxicity

1,4-BDDMA was studied in an OECD 422 combined repeat dose and reproductive/developmental toxicity screening test (RTC, 2013). Groups of 10 male and 10 female rats were administered by gavage at dose levels of 0, 100, 300, or 1000 mg/kg/day. Male rats were dosed for 33 days and female rats were dosed from 14 days prior to mating through Day 3 of lactation. On the basis of the results obtained in this study, there were significant signs of toxicity in the 1000 mg/kg/day group for males and females with histopathological findings in the liver (females) and stomach (males and females). A reduction of the fertility index has been observed at 1000 mg/kg/day. The NOAEL for effects on fertility as well as for parental toxicity was found to be 300 mg/kg/day.

No further reproduction toxicity tests are available with 1,4-Butanediol dimethacrylate.

1,4-Butanediol dimethacrylate will rapidly be hydrolyzed by unspecific carboxylesterases in the liver into methacrylic acid and 1,4 -butanediol (see chapter Toxikokinetics and the Category document, respectively).

Therefore corresponding to the requirements of Annex X higher studies were covered in a read across approach with the hydrolyses products methacrylic acid plus its metabolite donor substance methyl methacrylate, 1,4-Butanediol plus its donor substance Gamma-Butyrolactone and the analogous substance ethylene glycol. Animal testing with Methyl methacrylate avoids the local high toxicity of MMA due to its acidity.

1,4-Butanediol

In a developmental toxicity study with Swiss CD-1 mice according NTP protocol animals recieved 100, 300 or 600 mg/kg/day 1,4-Butanediol from gd 6 to gd 15. In summary, 100 mg/kg/day 1,4-butanediol was a no-observed-adverse-effect-level (NOAEL) for maternal and developmental toxicity. Dams exposed to 300 or 600 mg/kg/day exhibited sedation, decreased food intake during and after treatment and decreased body weight gain (92 and 83 % of control weight,respectively). A trend toward decreased late fetal death was observed, but the cumulative incidence of post-implantation mortality (resorptions plus late fetal deaths) was not altered by exposure to 1,4-butanediol. Developmental toxicity was expressed primarily as decreased fetal body weight at 300 and 600 mg/kg/day ,with a trend toward increased skeletal defects at 600 mg/kg/day (Price 1993).

Gamma-butyrolactone

In a teratogenicity study similar to OECD 414 with a reduced number of animals γ-Butyrolactone was tested in Sprague Dawley-rats at concentrations of 10, 50, 125, 250 and 500 mg/kg/day. Maternal toxicity was observed for mean fetal weights and placental weights. As no dose realtionship was observed clear toxicity could not be identified. No toxicity was observed on the offspring at the highest dose tested. Therefore it can be concluded that γ-Butyrolactone is not teratogenic at concentrations up to 500 mg/kg/d. NOAEL for developmental toxicity is 500 mg/kg/d (Kronevi 1998).

Ethylene glycol

The developmental toxicity of ethylene glycol in animals has been assessed by several animal studies. From those, oral studies are considered most relevant for the metabolite of EGDMA. In brief, developmental effects in animal studies have been shown to be species specific and, in addition are effected by the dosing regime. For REACh regulation reasons, a study with a non-rodent species is of most relevance for the alcohol metabolite pathway (Tyl 1993). As stated by NTP-CERHR (2004), “rabbits demonstrated no developmental toxicity following gavage exposure to doses as high as 2,000 mg/kg bw/day on gd 6–19, as noted by a lack of malformations, prenatal deaths, or decrease in fetal weights. Severe maternal toxicity was observed at 2,000 mg/kg bw/day as evidenced by maternal deaths, increased early delivery, and lesions as well as oxalate crystals in the kidneys. Maternal and fetal NOAELs were identified as 1,000 and 2,000 mg/kg bw/ day, respectively. Thus, the data were sufficient to demonstrate a lack of developmental toxicity in rabbits following oral gavage throughout organogenesis at doses ≤ 2,000 mg/kg bw/day.” More details are available in the category document.

Methacrylic acid

Methacrylic acid (MAA), the common metabolite for all the esters, also was tested in groups of 19-25 pregnant female rats (whole-body inhalation exposure for 6 hr/day, during days 6 to 20 of gestation), at 0, 50, 100, 200, and 300 ppm (0, 179, 358, 716 and 1076 mg/m3) and produced no embryo- or fetal lethality, nor fetal malformations after exposure with MAA at any concentration, despite overt maternal toxicity (decreased body weight and feed consumption) at 300 ppm (1076 mg/m3). The NOAEL for developmental toxicity was considered 300 ppm (1076 mg/m3) MAA (Saillenfait et al., 1999).

Methyl methacrylate

In a developmental toxicity study according to OECD 414 with Crl:CDBR rats, MMA was administered by inhalation exposure to 99, 304, 1178, and 2028 ppm (412, 1285, 4900, 8436 mg/m³; Rohm and Haas, 1991).Treatment related maternal effects on body weight and food consumption were noted at all exposure levels, consequently the LOEC for maternal toxicity is 99 ppm. No embryo of foetal toxicity was evident and no increase in the incidence in the malformations or variations was noted at exposure levels up to and including 2028 ppm. In this study the NOAEC for developmental toxicity was 2028 ppm (8436 mg/m³).

In addition, another study with MMA has been performed, an oral OECD 414 study in rabbits at 50, 150 , and 405 mg/kg/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 450 mg/kg bw/d. No adverse foetal findings of toxicological relevance were evident at any dose, even in the presence of maternal toxicity (body weight and food consumption effects at 150 and 405 mg/kg/d; BASF, 2009). MMA is not a selective teratogen.

Based on the available data on 1,4 -Butanediol dimethacrylate or its metabolites 1,4 -Butanediol plus Gamma-Butyrolactone/ Ethylene Glycol and Methacrylic acid plus Methyl Methacrylate in rodents and non-rodents 1,4 -Butanediol dimethacrylate is not considered to cause prenatal developmental toxicity.

Compliance to REACh requirements

The screening study requirement is covered with a reliable OECD 422 oral rat study, performed with the substance itself. The development toxicity requirements for two species are covered with reliable oral or inhalation studies with the methacrylic metabolite MAA, its donor MMA, or with the alcohol metabolite 1,4-BD, its donor γ-BL, or the metabolite analogue and EG. The read across is done with a high level of confidence (see chapter Toxicokinetics and the Category Document, respectively)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Remarks:
screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 20 April to 25 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
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
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.
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.
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.
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
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
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
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
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified
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, 9in 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.
No changes of toxicological significance were found.
The main relevant change was an increased value of bile acids in treated groups compared tocontrols 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 markedlyreduced in the high dose group (40% compared to 90% of the control group).
No significant differences were observed in the number of implantation, 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.
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.
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.
No treatment-related changes were noted at macroscopic examination.
Treatment-related findings at microscpopic 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.Stomach (non-glandular)
The treatment-related change seen in the high dosed animals (1/10 and 5/10, respectively inmales 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.
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.
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 and reproduction/developmental toxicity could be considered 300 mg/kg/day for males and females. 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.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
NTP test guideline
Justification for type of information:
Read across from the alcohol metabolite.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1ff

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, table in chapter 1.2 and endpoint specific chapters
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: RTI Master Protocol No. 360/NTP Protocol No. NTP-90-CERT-133
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
CD-1
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Timed-mated Swiss albino (CD-1) mice were given 1,4-butanediol in aqueous solution on gestional days 6 through 15.
Duration of treatment / exposure:
gestational day 6 to 15
Frequency of treatment:
daily
Duration of test:
until gd 17
No. of animals per sex per dose:
28-32 per dose group
Control animals:
yes, concurrent no treatment
Maternal examinations:
Maternal body weights, food and water consumption, and clinical signs were monitored at regular intervals throughout gestation.
Ovaries and uterine content:
At termination (gd 17), the number of resorptions, and dead or live fetuses were recorded.
Fetal examinations:
Live or dead fetuses were weighed. Live fetuses were sexed and examined for external, visceral and skeletal malformations
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Other expressions of maternal toxicity included significant reductions of body weight on gd 12 and 17 (mid dose) or gd 9-17 (high dose). Maternal body weight gains were significantly reduced in the mid- and high-dose groups as follows: weight gain during treatment (gd 6 to 15), weight gain during gestation (gd 0 to 17), and gestational weight gain corrected for gravid uterine weight.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mid and high-dose dams also showed significantly reduced food intake (g/kg/day) throughout both the treatment period (gd 6 to 15) and the post-treatment period (gd 15 to 17).
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Hypoactivity, immobility, loss of righting reflex, and/or prone posture were observed after daily administration of 300 or 600 mg/kg/day. Symptoms of intoxication resolved within 4 hours after each treatment and tolerance did not appear to develop across ten days of administration.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute maternal liver weight was decreased at the mid- and high-doses and absolute maternal kidney weight was decreased at the high dose. Relative maternal liver and kidney weights (% body weight on gd 17) were unaffected by 1,4-butanediol exposure.
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The cumulative incidence of post-implantation mortality (resorptions plus late fetal deaths) was not altered by exposure to 1,4-butanediol.
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
A decreasing trend was observed for the percent late fetal deaths per litter, and the percentage of litters with one or more late fetal deaths was actually lower at 300 and 600 mg/kg/day
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No maternal deaths occurred in this study. The low dose (100 mg/kg/day ) produced no significant maternal effects. In contrast, acute symptoms of intoxication (hypoactivity, immobility, loss of righting reflex, and/or prone posture) were observed after daily administration of 300 or 600 mg/kg/day 1,4-butanediol. Symptoms of intoxication resolved within 4 hours after each treatment. and tolerance did not appear to develop across ten days of treatment. Other expressions of maternal toxicity included significant reductions of body weight on gd 12 and 17 (mid dose) or gd 9-17 (high dose). Maternal body weight gains were significantly reduced in the mid- and high-dose groups as follows: weight gain during treatment (gd 6 to 15), weight gain during gestation (gd 0 to 17), and gestational weight gain corrected for gravid uterine weight. Mid and high-dose dams also showed significantly reduced food intake (g/kg/day) throughout both the treatment period (gd 6 to 15) and the post-treatment period (gd 15 to 17). Maternal water intake was not affected. Absolute maternal liver weight was decreased at the mid- and high-doses and absolute maternal kidney weight was decreased at the high dose. Relative maternal liver and kidney weights (% body weight on gd 17) were unaffected by 1,4-butanediol exposure.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Developmental toxicity was expressed primarily as a significant reduction in live fetal body weight per litter (92% and 83% of controls in the mid and high-dose groups, respectively).
Developmental toxicity was expressed primarily as decreased fetal body weight at 300 and 600 mg/kg/day
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): Developmental toxicity was expressed primarily as a significant reduction in live fetal body weight per litter (92% and 83% of controls in the mid and high-dose groups, respectively).
Changes in sex ratio:
not specified
Changes in litter size and weights:
effects observed, non-treatment-related
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
An increasing trend was observed for the percent fetuses with skeletal malformations per litter and for the percentage of litters with one or more skeletally malformed fetuses. The most frequently observed skeletal defects observed in the high dose group were fused, missing or branched ribs and fused thoracic vertebrae (fused centra or arches).
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Developmental toxicity was expressed primarily as a significant reduction in live fetal body weight per litter (92% and 83% of controls in the mid and high-dose groups, respectively). While the incidence of resorptions was comparable across groups, a decreasing trend was observed for the percent late fetal deaths per litter, and the percentage of litters with one or more late fetal deaths was actually lower at 300 and 600 mg/kg/day . The incidence of fetuses with external (gross) or visceral malformations was comparable across groups, but an increasing trend was observed for the percent fetuses with skeletal malformations per litter and for the percentage of litters with one or more skeletally malformed fetuses. The most frequently observed skeletal defects observed in the high dose group were fused, missing or branched ribs and fused thoracic vertebrae (fused centra or arches).
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: rib
skeletal: vertebra
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Developmental toxicity observed only in presence of maternal toxic doses.

Executive summary:

In a developmental toxicity study with Swiss CD-1 mice according NTP protocol animal recieved 100, 300 or 600 mg/kg/day from gd 6 to gd 15. In summary, 100 mg/kg/day 1,4-butanediol was a no-observed-adverse-effect-level (NOAEL) for maternal and developmental toxicity. Dams exposed to 300 or 600 mg/kg/day exhibited sedation, decreased food intake during and after treatment and decreased body weight gain (92 and 83 % of control weight, respectively). A trend toward decreased late fetal death was observed, but the cumulative incidence of post-implantation mortality (resorptions plus late fetal deaths) was not altered by exposure to 1,4-butanediol. Developmental toxicity was expressed primarily as decreased fetal body weight at 300 and 600 mg/kg/day , with a trend toward increased skeletal defects at 600 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Read across from the alcohol metabolite donor substance.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1ff

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, table in chapter 1.2 and endpoint specific chapters
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Only 10 animals per dose used instead of 20 but 5 dose levels tested instead of 3
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
sexually mature nulliparous females (240-330g)
mature males
Sprague-Dawley albino rats (Anticimex AB, Sollentuna, Sweden)
Route of administration:
oral: gavage
Vehicle:
soya oil
Details on mating procedure:
The rats were mated (five females to one male) and the day sperm was found in the vaginal smear was considered to be day 0 of gestation. The females were then randomly allocated to individual cages and were individually coded.
Duration of treatment / exposure:
GBL was administered to rats by gavage on gestation days 6 through 15.
Frequency of treatment:
once daily
Duration of test:
10 days
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 (control: 9)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The highest amount of GBL used, 500 mg in 5 ml soy bean oil, was difficult to get dissolved. Higher amounts of GBL were thus not considered for testing.
Maternal examinations:
Each female was observed daily throughout the experimental period for physical condition and for signs of toxicity. Body weights were recorded daily from day 0 through day 21 of gestation. Food and water consumption was recorded for each rat at approximately 3-day intervals beginning on day 0.
Ovaries and uterine content:
On day 21 the females were anaesthetized by ethyl ether and the foetuses were removed by Caesarean section. Data on position and number of foetuses in utero, number of alive and dead foetuses, resorptions, and corpora lutea, the sex of each foetus and individual foetal weight, gross external anomalies, and placental weights were recorded.
Fetal examinations:
Body weight of fetus was observed. For each litter, preimplantation and postimplantation foetal losses were calculated as percentage.
Every third alive foetus of each litter was preserved in Bouin’s solution and later examined under a dissection microscope (6.4-40 x enlargement) for soft tissue anomalies after using free hand razor sectioning technique (Wilson 1965). The remaining foetuses were fixed in ethyl alcohol and cleared in 2% KOH stained with alizarin red S and stored in glycerin for later examination for skeletal defects.
Statistics:
The differences between exposed groups and control groups with respect to numbers of corpora lutea, implantation sites, alive foetuses per litter, preimplantation loss, postimplantation loss as well as mean foetal weight, maternal body weight, placental weight, water and food consumption, was calculated using Student’s t-test. The number of malformations was analyzed using Chi-square test with Yate’s correction.
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
placental weights were significantly reduced for treated animals
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
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:
no effects observed
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
placental weights were significantly reduced for treated animals
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Placental weights and mean fetal weights

Details on maternal toxic effects:
Placental weights were significant reduced for treated animals. The difference is similar in all dose groups compared to the control. No dose relationship is observed. Mean fetal weights are significantly increased compared to the control values in the 50, 125 and 250 mg/kg/d groups. No dose relationship is observed.

No significant differences were found between the control group and the treated groups as regards corpora lutea and total implantation sites, alive and dead foetuses, resorptions, preimplantation and postimplantation losses, or male/female ratios.
Dose descriptor:
LOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other:
Remarks:
no dose dependent effect
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean foetal weights were significantly increased compared to control in the 50, 125, 250 mg/kg/day groups
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Mean feotal weights were significantly increased compared to control values in the 50, 125, 250 mg/kg/day group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Gastrochisis in 1/2 animals in the 125/500 mg/kg dose group. The findings have been observed in this strain earlier in the test laboratory.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Examination of the foetuses for skeletal alterations revealed a slight (P=0.034) decrease in the incidence of bipartite centra of the thoraic vertebrae in the 500 mg/kg/day group and a slight increase in the frequency of unossified hyoid cartilage in the 10 and 125 mg/kg/day groups.
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
highest dose tested 500 mg/kg bw/day due to limitation of solubility in the vehicle
Abnormalities:
not specified
Developmental effects observed:
not specified

Summary of litter and reproduction data for pregnant rats fed γ-Butyrolactone.

Dose mg/kg b.wt. Control  10 50 125 250 500
No. of females in group 9 10 10 10 10 10
No. of females that died 0 0 0 1 10 10
Mean maternal body weight gain (g) day 103.0 +/-27.2 120.8 +/- 29.6 124.6 +/- 28.8 111.7 +/- 32.6 126.9 +/- 20.2 106.1 +/- 24.7
0-21 (+/- S.D.) a)            
No. of surviving pregnant females 9 7 9 9 9 7
No. of corpora lutea per dam 19.3 +/- 4.0 16.6 +/- 1.4 19. +/- 3.6 19.9 +/- 3.0 19.0 +/- 4.9 20.4 +/- 5.1
(Mean k +/- S.D.)            
No. of implantations sites per dam 11.2 +/- 4.3 13.7 +/- 3.6 13.5 +/- 2.9 12.8 +/- 3.1 14.1 +/- 2.7 11.9 +/-4.6
(Mean +/-S.D.)            
No. of live foetuses per litter 10.8 +/- 4.0 13.3 +/- 4.1 13.1 +/-2.8 12.1 +/-2.8  13.7 +/-2.7  11.1 +/-4.3 
(Mean +/- S.D.)
Total no. of resorptions 3 3 2 4 4 5
Total no. of dead foetuses 1 0 0 1 0 0
Preimplantation loss % 40.5 +/-26.5 17.8 +/- 19.8 29.7 +/- 18.5 32.2 +/- 14.0 24.5 +/- 17.2  40.7 +/-27.1
Postimplantation loss % 3.0 +/-4.6 5.65 +/- 11.5 1.9 +/- 3.3 4.7 +/-4.6 2.6 +/-4.9 4.4 +/-7.0
Male/female ratio 54/43 44/49 35/64 54/56 49/64 34/44
Mean fetal weight (+/- S.D.) 5.26 +/-0.49 5.29 +/- 0.57 5.53 +/- 0.61*** 5.52 +/- 0.39*** 5.44 +/- 0.56* 5.39 +/- 0.5 I
Placental weight (g) 0.47 +/- 0.10 0.44 +/- 0.08* 0.44 +/- 0.08  0.42 +/- 0.08***  0.44 +/- 0.07**  0.43 +/- 0.14* 

* P 0.05-0.01; ** P 0.014l.001; *** P<O.O01. a) Pregnant and surviving females only.

Incidence of foetal malformations (external, visceral and skeletal) in rats fed γ-Butyrolactone.

Dose mg/kg b.wt. Control  10 50 125 250 500
External malformations            
No. Examined 98 93 118 111 123 78
gastroschisis 0 0 0 1 (0.9%) 0 2 (2.6%)
Skeletal malformations            
No. Examined 68 65 80 77 85 56
Hyoid cartilage unossified 12 (17.6%) 22* (33.8%) 5 (31.2%) 24* (31.2%) 13 (15.3%) 10 (17.9%)
Bipartite cervical vertebrae 0 0 0 0 1(1.25%) 0
Rudiment on cervical vertebrae 2 (2.9%) 0 0 1 (1.35%) 5 (5.9%) 2 (3.6%)
Unossified or bipartite thorax vertebrae 32 (47.0%) 24 (36.9%) 41 (51.2%) 36 (46.8%) 29 (34.1%) 15* (26.8%)
Bipartite lumbar vertebrae 2 (2.9%) 0 1 (1.1%) 0 0 0
7 sternum vertebrae 0 0 7* (8.7%) 0 0 0
Assymetrical or bipartite - sternum vertebrae 12 (17.6%) 13 (20.0%) 20 (25.0%) 8 (10.4%) 18 (21.2%) 11 (19.6%)
13 ribs dexter 61 (89.7%) 55 (84.6%) 66 (82.5%) 64 (83.1%) 79 (92.9%) 48 (85.7%)
sinister 60 (88.2%) 55 (84.6%) 61 (76.2%) 59 (76.6%) 75 (88.3%) 45 (80.4%)
14 ribs dexter 7 (10.3%) 10 (15.4%) 13 (16.2%) 13 (16.9%) 6 (7.1%) 7 (12.5%)
sinister 8 (11.8%) 10 (13.7%) 18 (22.5%) 18 (23.4%) 10 (11.8%) I 1 (19.6%)
Animals with 15 ribs 0 0 7* (8.7%) 0 0 0
Internal soft tissue malformations            
No. examined 30 28 38 34 38 22
Malformations 0 0 0 0 0 0

* P 0.05-0.01

Conclusions:
In a teratogenicit study with γ-Butyrolactone in rats in concentrations of 10, 50, 125, 250 and 500 mg/kg/day effects were observed for mean fetal weight and placental weight. As no dose realtionship was observed clear toxicity cannot be identified. No toxicity was observed on the offspring.
Therefore it can be concluded that γ-Butyrolactone is not teratogenic at concentrations up to 500 mg/kg/d. NOAEL for developmental toxicity is 500 mg/kg/d
Executive summary:

In a teratogenicity study similar to OECD 414 with a reduced number of animals γ-Butyrolactone was tested in Sprague Dawley-rats at concentrations of 10, 50, 125, 250 and 500 mg/kg/day. Effects were observed for mean fetal weights and placental weights. As no dose realtionship was observed clear toxicity could not be identified. No toxicity was observed on the offspring at the highest dose tested.

Therefore it can be concluded that γ-Butyrolactone is not teratogenic at concentrations up to 500 mg/kg/d. NOAEL for developmental toxicity is 500 mg/kg/d.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
DATA QUALITY: Study was conducted in accordance with a recognized scientific procedure for determining the developmental toxicity of a test substance when administered repeatedly via inhalation. Study was conducted incompliance with GLP regulations. The study meets national and international scientific standards (OECD 414) and provides sufficient information to support the conclusions regarding the NOAEL and the LOAEL demonstrated from the study data.
Justification for type of information:
Read across from the methacrylic metabolite
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1ff
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMP
URITIES)
see attached category document, chapter 1
3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters
4. DATA MATRIX
see attached category document, table in chapter 1.2 and endpoint specific chapters
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Nulliparous female Sprague-Dawley rats, weighing 180-200  grams.obtained from IFFA Credo Breeding Labs. AGE at Start of Test: sexually mature females; age not specified. Mated females were  housed inclear polycarbonate cages with stainless steel wire lids and  hardwood shavings for bedding. Food and water available adlibitum except  during exposures.
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
 Exposures were whole  body and conducted in a 200 L chamber. Chamber temperature was 23 degrees  C, and the relative humidity was 50%. Air was passed through a heated  bubbler containing test material. The vaporized material was then  introduced into the exposure chambers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
 Concentrations were monitored  continuously with a GC, and were determined once during each 6 hr  exposure by collecting the material and analyzing against a standard  using GC.   
Details on mating procedure:
2-3 females were caged with one male rat for mating. The  onset of gestation was based upon the presence of sperm in the vaginal  smear and this was designated gestation day 0. After confirmation of  mating, females werere turned to an individual cage. 
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
day 6 to 20 of gestation
Duration of test:
Mated females were exposed 6 hr/day on days 6 through  20 of gestation.
Dose / conc.:
50 ppm
Remarks:
corresponds to 179 mg/m3
Dose / conc.:
100 ppm
Remarks:
corresponds to 358 mg/m3
Dose / conc.:
200 ppm
Remarks:
corresponds to 716 mg/m3
Dose / conc.:
300 ppm
Remarks:
corresponds to 1076 mg/m3
No. of animals per sex per dose:
22-25 pregnant females per dose.
Control animals:
other: yes, concurrently to filtered room air
Ovaries and uterine content:
 The uterus was removed and weighed. At necropsy, the uterine horns and ovaries were exposed  to count the C.L., implantation sites, resorption sites, and viable and  dead fetuses.   FERTILITY AND REPRODUCTIVE PERFORMANCE: The following data were recorded  for each group of numbers of CL, and implantation sites o number of  resorptions and viable and dead fetuses. O mean fetal body weights o  fetuses examined for gross malformations and skeletal abnormalities of  sex and of fetuses.
Fetal examinations:
Live fetuses were weighed, sexed, and examined for external  anomalies. 50% of the live fetuses were preserved in Bouin's solutionand  examined for internal soft-tissue changes. The remaining fetuses were  fixed in ethanol (70%), eviscerated and then processed for skeletal  staining with alizarin red S.
Statistics:
 The number of CL, implantation sites,and live  fetuses, maternal food consumption and various body weights were analyzed  by ANOVA, followed by Dunnett'st-test. the percentage of non-live  implant, resorptions,and males and the proportion of fetuses with  alterations ineach litter were evaluated by Kruskal-Walles test followed  by Dixon-Massey test. Rates of pregnancy and percentage of litters with  any malformations or external, visceral, or skeletal variations were  analyzed using Fisher's test. Where appropriate, least squares analysis  was performed. The level of significance was p < 0.05.
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Absolute weight gain was significantly reduced at 300 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Fopod consumption was reduced at 300 ppm.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
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:
no effects observed
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All animals survived the exposure. Exposure to 300 ppm led to significant decreases in maternal weight gain and food consumption throughout exposure. Absolute weight gain was significantly reduced at 300 ppm.
Dose descriptor:
NOAEL
Effect level:
200 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no significant changes in number of implantations and live fetuses, in the incidence of non-live implants and sorptions, or in fetal weights across groups. One fetus of 200 ppm and two of the 300 ppm group showed different types of malfomations. There was no consistent pattern of changes to suggest any treatment-related effects. The difference of fetuses with external, visceral, and skeletal variations did not differ between the control and the treated groups. No significant increase in embryo/fetal lethality or fetal malformations were observed after exposure to methacrylic acid. While maternal toxicity was observed, methacrylic acid caused no  evidence of developmental toxicity up to 300 ppm.
Dose descriptor:
NOAEL
Effect level:
>= 300 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
Using a valid scientific method, no significant increase in embryo/fetal lethality or fetal malformations were observed after exposure to methacrylic acid. While maternal toxicity was observed, methacrylic acid caused no  evidence of developmental toxicity up to 300 ppm.
Executive summary:

In an OECD 414 prenatal developmental toxicity study using whole body inhalation methacrylic acid produced no embryo - or foetal lethality, nor fetal malformations after exposure with methacrylic acid, despite overt maternal toxicity (decreased body weight and feed consumption). The NOEC (teratogenicity) was considerd to be 300 ppm (1076 mg/m³).

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

 

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Although the study was only reported in 2009 it was already commisioned before the establishment of ECHA and therefore no test proposal could be submitted.
Justification for type of information:
Read across from the methacrylic metabolite donor substance.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1ff

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, table in chapter 1.2 and endpoint specific chapters
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
Himalayan
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 16-21 weeks
- Weight at study initiation: 2187-2917 g
- Fasting period before study: no
- Housing: the rabbits were housed singly in type 12.2395.C stainless steel wire mesh cages supplied by Draht-Bremer GmbH, Marktheidenfeld, Germany (floor area about 3,000 cm²)
- Diet: pelleted “Kliba maintenance diet for rabbits & guinea pigs, GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. to 6.00 a.m. dark, 6.00 a.m. to 6.00 p.m. light)
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at maximum intervals of 7 days, which took into account the analytical results of the stability verification. For the test substance preparation, an specific amount of test substance was weighed depending on the dose group, into a graduated flask (conical Erlenmeyer flasks with groundin stopper), topped up (shortly under the marking) with 1% Carboxymethylcellulose solution in drinking water and a few drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The flask was sealed and the preparation was intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer and the vessels were kept closed between the withdrawals of the preparations.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. Samples were analyzed by GC with external calibration.
Details on mating procedure:
After an acclimatization period of at least 5 days, the female rabbits were fertilized by means of artificial insemination. This implied that 0.2 mL of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe (Receptal) were injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study. The day of insemination was designated as gestation day (GD) 0 and the following day as GD 1.
Duration of treatment / exposure:
from implantation to one day prior to the expected day of parturition (GD 6-28)
Frequency of treatment:
daily
Duration of test:
until GD 29
No. of animals per sex per dose:
25 inseminated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose volume was 10 mL/kg body weight. The calculation of the volume administered was based on the most recent individual body weight.
Maternal examinations:
CLINICAL EXAMINATIONS
- Mortality: Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).
- Clinical symptoms: A clinical examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-29).
- Food consumption: The food consumption was determined daily on GD 1–29.
- Body weight data: All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated.
- Corrected (net) body weight gain: The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

TERMINAL EXAMINATIONS OF THE DOES
After the does had been sacrificed on GD 29, they were necropsied and assessed by gross pathology in randomized order.
Ovaries and uterine content:
On GD 29, the surviving does were sacrificed in randomized order by an intravenous injection of pentobarbital (Narcoren; dose: 2 mL/animal). After the does had been sacrificed, they were necropsied and assessed by gross pathology in randomized order. The uterus and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
1) live fetuses
2) dead implantations: a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy); b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible); c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).
After the weight of the uterus had been determined, all subsequent evaluations of the does and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose the animal numbers were encoded.
Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out:
The conception rate (in %) was calculated according to the following formula: (number of pregnant animals)/(number of fertilized animals) x 100.
The preimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice according to the following formula: (number of corpora lutea – number of implantations)/(number of corpora lutea) x 100.
The postimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice from the following formula: (number of implantations – number of live fetuses)/(number of implantations) x 100.
Fetal examinations:
All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.
- Examination of the fetuses after dissection from the uterus: Each fetus was weighed and examined macroscopically for any external findings.
Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes, and fluids were examined. Individual placental weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of phenobarbital (Narcoren; 0.2 mL/fetus).
- Soft tissue examination of the fetuses: After the fetuses had been sacrificed, the abdomen and the thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure. The sex of the fetuses was determined by examination of the gonads in situ. After these examinations, the heads of approximately one half of the fetuses per litter and the heads of those fetuses, which revealed severe findings during the external examination (e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN's solution and were, after fixation, processed and evaluated according to WILSON's method (Wilson and Warkany, 1965). About 10 transverse sections were prepared per head. After the examination these heads were discarded. All fetuses (partly without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the fetuses were removed from the fixative for awhile. With a scalpel, a transversal incision was made into the frontal / parietal bone in the heads of the intact fetuses. The two halves of the calvarium were then cauteously bent outward and the brain was thoroughly examined. Subsequently, the fetuses were placed back into the fixative for further fixation.
- Skeletal examination of the fetuses: After fixation in ethyl alcohol the skeletons were stained according to a modified method of KIMMEL and TRAMMELL (Kimmel, C.A., and Trammell, C., 1981). Thereafter, the stained skeleton of each fetus was examined. After the examination the stained skeletons were retained individually.
Statistics:
- DUNNETT-test (two-sided) for food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight.
- FISHER'S EXACT test (one-sided) for female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings.
- WILCOXON-test (one-sided) for proportions of fetuses with malformations, variations and/or unclassified observations in each litter.
Details on maternal toxic effects:
Details on maternal toxic effects:
- Mortality: There were no test substance-related or spontaneous mortalities in any group.
- Clinical symptoms: No test substance-related clinical signs or any disturbances of the general behavior were observed in any rabbit during the entire study period.
- Food consumption: The food consumption in the high-dose females (450 mg/kg bw/d) was distinctly and statistically significantly reduced during a significant part of the treatment period (GD 15-23). During the entire treatment period (GD 6-28) the total average food consumption of the high dose rabbits was about 18% below controls. The food consumption of the mid dose females (150 mg/kg bw/d) was similarly affected in terms of magnitude and course of reduction, however the reduction of food consumption reached statistical significance only on GD 22-24. During the treatment period (GD 6-28) the total average food consumption of the mid-dose rabbits was about 13% below controls. Overall, the food consumption of the low-dose does (50 mg/kg bw/d) did not show test substance-related impairments. The reduced food consumption at the 150 and 450 mg/kg bw/d levels is considered to be related to the treatment.
- Body weight data: The mean body weights of the low-, mid- and high-dose rabbits (50; 150 and 450 mg/kg bw/d) were not significantly different from the concurrent control throughout the course of the study. The average body weight gain of the mid- and high-dose rabbits was statistically significantly reduced by about 27% and 31% during the treatment period. A significant reduction of mean body weight gain was also noted for the the high-dose rabbits on GD 19-21.
- Corrected (net) body weight gain: Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were comparable among all groups.
- Uterus weight: The mean gravid uterus weights of test groups 1, 2, and 3 (50; 150 or 450 mg/kg bw/d) did not show statistically significant differences in comparison to the control group.
- Necropsy findings: At necropsy, only spontaneous findings were seen in single females of every test group. No test substance-related findings were observed in the does.
- Reproduction data of does: The conception rate reached 96% in test groups 1 and 3 (50 and 450 mg/kg bw/d) and 100% in test groups 0 and 2 (0 and 150 mg/kg bw/d). Importantly, a sufficient number of pregnant females was available for the purpose of the study, as 24-25 pregnant rabbits per group had implantation sites in the uterus, at terminal sacrifice. There were no test substance-related and/or biologically relevant differences between the control and all dosed groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Gestational parameters were within the normal range for animals of this strain and age.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
- Sex distribution of fetuses: The sex distribution of the fetuses in test groups 1-3 (50; 150 and 450 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.
- Weight of placentae: The mean placental weights in test groups 1, 2 and 3 (50; 150 and 450 mg/kg bw/d) were comparable to the controls.
- Weight of fetuses: The mean fetal weights of all treated groups were not influenced by the test substance. Neither female nor male weights showed statistically significant or biologically relevant differences between the test substance-treated groups and the controls.
- Fetal external malformations: One sole external malformation (unilateral microphthalmia) was recorded for two fetuses from 2 litters in the high-dose group (450 mg/kg bw/d). This malformation is present in the historical control data. Thus an association of these individual findings to the treatment is not assumed. The total incidences of external malformations were comparable to the historical control data.
- Fetal external variations: One external variation (paw hyperflexion) occurred in single fetuses of the low- and mid-dose groups and the control. The incidences did not demonstrate a dose-response relationship and were comparable to the historical control data. Thus an association of this finding to the treatment is not assumed.
- Fetal external unclassified observations: Unclassified external observations, such as necrobiotic placentae and discolored amniotic fluid, were recorded for single fetuses of test groups 1 and 2 (50 and 150 mg/kg bw/d). A relation to dosing is not present if normal biological variation is taken into account. Therefore, a test substance induced effect is not assumed.
- Fetal soft tissue malformations: The examination of the soft tissues revealed a variety of malformations in fetuses of all test groups including the controls (0; 50; 150 and 450 mg/kg bw/d). With the exception of a lateral pouch in the tongue of 2 fetuses all individual soft tissue malformations were present in the historical control data at comparable frequencies. No statistically significant differences between the test groups and the control were observed. The total incidences of external malformations were comparable to the historical control data. No malformation pattern was evident. Thus an association of these findings to the treatment is not assumed.
- Fetal soft tissue variations: A number of soft tissue variations, such as absent lung lobe (lobus inferior medialis) and malpositioned carotid branch, was detected in each test group including the controls. Incidences were without a relation to dosing. Neither statistically significant differences between the test groups nor differences to the historical control data were noted.
- Fetal soft tissue unclassified observations: Unclassified soft tissue observations, such as infarct of liver, hemorrhagic thymus or ovary and blood coagulum around urinary bladder, were recorded for some fetuses of test groups 0, 1, 2 and 3 (0; 50; 150 and 450 mg/kg bw/d). A relation to dosing is not present if normal biological variation is taken into account. Therefore, a test substance induced effect is not assumed.
- Fetal skeletal malformations: Malformations of the fetal skeletons were noted in fetuses of test groups 0, 2 and 3 (0; 150 and 450 mg/kg bw/d). Neither statistically significant differences between treated groups and the control were calculated nor a dose-response relationship was observed. All individual skeletal malformations were present in the historical control data at a comparable frequency.
- Fetal skeletal variations: For all test groups, variations in different skeletal structures were detected with or without effects on the corresponding cartilages. The observed skeletal variations were related to various parts of the fetal skeletons and were statistically significant higher in the low- and the
high-dose groups on a fetus per litter basis. Several specific skeletal variations were statistically significant higher than the concurrent control in the dosed groups (on a fetus per litter basis). These findings are delays or minor disturbances of ossification which are reversible or do not considerably affect the integrity of the underlying structures. Such slight changes of the ossification process occur very frequently in gestation day 29 rabbit fetuses of this strain and all observed incidences were within the historical control data. Thus an association of these findings to the treatment is not assumed.
- Fetal skeletal unclassified cartilage observations: Additionally, isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings did not show a relation to dosing and were comparable to historical control data and, therefore, regarded to be spontaneous in nature.
- Abstract of all classified fetal external, soft tissue and skeletal observations: Various external, soft tissue and skeletal malformations occurred throughout all test groups including the control. All individual malformations are present in the historical control data, with the exception of lateral pouches in the tongue of 2 fetuses. They did neither show a consistent pattern since a number of morphological structures of different ontogenic origin were affected nor a clear dose-response relationship. The overall incidence of malformations was comparable to the historical control data. One external (paw hyperflexion), two soft tissue (absent lobus inferior medialis and malpositioned carotid branch) and a broad range of skeletal variations occurred in all test groups including the controls. All fetal and litter incidences for these variations and the corresponding mean percentages of affected fetuses/litter were not significantly different from the concurrent control and their frequency is comparable to the historical control data. Therefore, they were not considered to be related to the treatment. A spontaneous origin is also assumed for external, soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups including controls (0, 50; 150 and 450 mg/kg bw/d). Distribution and type of these findings do not suggest relation to treatment.
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other:
Remarks on result:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table: Occurence of statistically significantly increased fetal skeletal variation (expressed as mean percentage of affected fetuses/litter)

Finding

Group 0

0 mg/kg/d

Group 1

50 mg/kg/d

Group 2

150 mg/kg/d

Group 3

450 mg/kg/d

HCD

(range)

Incomplete ossification of parietal; unchanged cartilage

0.0

0.0

1.9*

2.1*

0.4

(0.0 – 2.6)

Incomplete ossification of hyoid; cartilage present

11.2

11.4

19.1

20.4*

9.8

(0.0 – 21.6)

Splitting of skull bone

0.4

3.3*

3.3

2.3

2.9

(0.0 – 7.7)

Incomplete ossification of cervical centrum; unchanged cartilage

2.5

2.2

3.6

7.3*

2.5

(0.0 – 9.3)

Supemumerary 13th rib; cartilage not present

2.5

9.8

6.1

9.9*

6.6

(0.0 – 17.5)

Total fetal skeletal variations

46.3

63.7*

59.3

71.6**

63.5

(46.3 – 81.9)

HCD = Historical control data; * = p ≤ 0.05, ** = p ≤ 0.01 (Wilcoxon-Test [one-sided])

 

 

Table: Total fetal malformations

 

 

Group 0

0 mg/kg/d

Group 1

100 mg/kg/d

Group 2

300 mg/kg/d

Group 3

1000 mg/kg/d

Litter

Fetuses

N

N

25

171

24

154

25

157

24

158

Fetal incidence

N (%)

4 (2.3%)

2 (1.3%)

6 (3.8%)

9 (5.7%)

Litter incidence

N (%)

4 (16%)

1 (4.2%)

4 (16%)

7 (29%)

Affected fetuses/litter

Mean%

2.3

1.2

3.6

6.2

 

 

Table: Total fetal variations

 

 

Group 0

0 mg/kg/d

Group 1

100 mg/kg/d

Group 2

300 mg/kg/d

Group 3

1000 mg/kg/d

Litter

Fetuses

N

N

25

171

24

154

25

157

24

158

Fetal incidence

N (%)

106 (62%)

106 (69%)

106 (68%)

122 (77%)

Litter incidence

N (%)

21 (84%)

24 (100%)

24 (96%)

23 (96%)

Affected fetuses/litter

Mean%

59.9

69.8

64.3

74.2

 

Conclusions:
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 50 mg/kg bw/d. The NOAEL for prenatal developmental toxicity is
450 mg/kg bw/d. No adverse fetal findings of toxicological relevance were evident at any dose.
Executive summary:

The study was performed according to OECD TG 414 in compliance with GLP.

Methyl Methacrylate was tested for its prenatal developmental toxicity in Himalayan rabbits. The test substance was administered as an aqueous preparation to 25 inseminated female Himalayan rabbits by stomach tube at doses of 50; 150 and 450 mg/kg body weight/day on gestation days (GD) 6 through GD 28. The control group, consisting of 25 females, was dosed with the vehicle (1% Carboxymethylcellulose CB 30.000 in drinking water and a few drops Cremophor EL and one drop hydrochloric acid [1% CMC]) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 29, 24-25 females per group had implantation sites.

The following test substance-related adverse effects/findings were noted:

Test group 3 (450 mg/kg body weight/day):

-        Reduced food consumption (-18%) and body weight gain (-31%)

-        No test substance-related adverse effects on gestational parameters or fetuses

 

Test group 2 (150 mg/kg body weight/day):

-        Reduced food consumption (-13%) and body weight gain (-27%)

-        No test substance-related adverse effects on gestational parameters or fetuses

 

Test group 1 (50 mg/kg body weight/day):

-        No test substance-related adverse effects on does, gestational parameters or fetuses

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 50 mg/kg bw/d. The NOAEL for prenatal developmental toxicity is 450 mg/kg bw/d. No adverse fetal findings of toxicological relevance were evident at any dose.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Method and result sufficient described, similar to OECD-guideline 414, GLP.
Justification for type of information:
Read across from the methacrylic metabolite donor substance.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1ff

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, table in chapter 1.2 and endpoint specific chapters
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CDBR
Details on test animals or test system and environmental conditions:
Nulliparous female rats, weighing 183-240 grams upon arrival.
AGE AT TIME OF MATING: 88-95 days. 
ACCLIMATION PRIOR TO MATING: 7 days 
SOURCE: Charles River Breeding Laboratories Inc., Kingston, NY.
Animals were housed individually, except during mating, in suspended stainless-steel cages (7" x 8" x 13.5"). During exposures, females were housed individually in suspended stainless-steel, wire mesh cages (6" x 7" x 11"). Temperature range was 23 ± 2°C and the relative humidity ranged from 40-60% during cohabitation and 63-80% during the exposure and post-exposure periods. Food (Certified Purina Rodent Chow #5002) and filtered tap water were available ad libitum except during exposures. A photoperiod of 12 hrs dark/ 12 hrs light was maintained.
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test material exposure concentrations were generated by metering the test material with calibrated Fluid Metering Pumps (Fluid Matering Inc., Oyster Bay, NY) into 500 mL three-necked round bottom flasks (Lab Glass Inc., Vineland, NJ).
Exposures were whole body and were conducted in 2000 L stainless steel, glass and Plexiglas® chambers. Cage positions within the chamber were rotated daily. The temperature and relative humidity within the chambers during exposure were 22-24°C and 55-67%, respective.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in the chambers was determined by the use of a Miran gas analyzer attached to a strip chart recorder. A probe was placed into the center of the chamber and the chamber atmosphere was drawn into the Miran A1 gas analyzer at a rate of 9.5 L/min. Each chamber was analyzed initially within 40 min. of the end of the t99 to insure that each chamber was within the accepted target range. Subsequently, each chamber was sampled every 120 min. A range of  plus or minus 10% of the target chamber concentration was maintained by making minor adjustments on the generator pump delivery rates whenever necessary.
Details on mating procedure:
Females were mated with males overnight (one male:one female) and the presence of sperm in the vaginal smear was considered  gestation day 0. Mated females were exposed via inhalation to the test material for 6 hrs/day on gestation days 6 through 15 and then sacrificed on day 20.  
Duration of treatment / exposure:
6 - 15 day of gestation
Frequency of treatment:
6 hours/day
Duration of test:
20 d (dams were euthanized on gestation day 20)
No. of animals per sex per dose:
27 animals per group exposed; 22-25 pregnant  females per exposure group.
Control animals:
yes, sham-exposed
Details on study design:
- Other: The strain was selected because background development toxicity data exists as Rohm and Haas Company on this rat strain. The test material was given by inhalation since the respiratory route is a potential route of human exposure.
Maternal examinations:
Maternal body weights were recorded on GD 0, 6, 8, 10, 13, 16 and 20. Food consumption was measured for GD intervals 0-6, 6-10, 10-16 and 16-20. Animals were observed daily for behavioral changes.
Ovaries and uterine content:
On GD 20, all dams were asphyxiated with carbon dioxide, the thoracic and abdominal cavities were examined and the uterus was removed and weighed, and corpora lutea, implantation sites and resorptions were counted. The number of fetuses per litter was counted and position inside the uterus recorded. The uteri of apparently non pregnant rats were stained with a 10% ammonium sulfide solution to detect very early resorptions. All fetuses were weighed, examined for external alterations and the sex of each fetus was determined. 
Fetal examinations:
One half of the fetuses from each litter were examined for visceral alterations using the Staples' technique. Head alterations were recorded for these fetuses examined for soft tissue alterations using the technique of Barrow and Taylor (1969, J. Morphol., 127: 291-306). The carcasses of all fetuses  were stained with alizarin red S and examined for skeletal alterations. 
Statistics:
For the purpose of statistical evaluation, the litter was considered the experimental unit for fetal parameters. Pregnancy rate, clinical signs, maternal deaths, gross necropsy findings  and liters with total resorptions were statistically analyzed using the  Fisher's exact test. Maternal body weight data and feed consumption values were statistically analyzed using Dunnett's test when the one-way ANOVA was significant. The number of implantations, live fetuses, resorptions, corpora lutea, mean fetal body weight/litter, and incidence of fetal alterations were statistically analyzed using the Mann-Whitney U test. When more than 75% ties occurred, then Fisher's exact test was used in place of the Mann-Whitney U test to detect significant differences between groups.
Details on maternal toxic effects:
Details on maternal toxic effects:
No animals died and no treatment-related clinical signs were noted for the dams in the 99, 304 or 1178 ppm groups. Scant feces was noted in the 2028 ppm group throughout the exposure period (GD 6-15). Treatment-related decreases on maternal body weight and feed consumption were noted at all exposure levels. The decreases in maternal body weight at 99 and 304 ppm were minimal and transient since they occurred only during the first 2 days of exposure and returned to control values by the  next weighing period. The body weight and feed consumption values returned to control values for all groups during the post exposure period  (GD 16-20). At 1178 and 2028 ppm, treatment-related effects included losses in maternal body and/or decreased body weight gain throughout the exposure period (GD 6 - 16) and decreased corrected maternal body weight gain. The gross necropsy evaluations did not indicate any treatment-related effects and there were no treatment-related differences between the control and treated groups in any reproductive parameter.  
Dose descriptor:
LOEC
Effect level:
ca. 0.41 mg/L air (analytical)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
 Fetal body weight was not affected by exposure to MMA vapors. The fetal external, visceral and skeletal examinations did not show any treatment related effects.
Dose descriptor:
NOAEC
Effect level:
>= 8.3 mg/L air (analytical)
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEC
Effect level:
>= 8.3 mg/L air (analytical)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Mean measured concentrations (± SD) within the chambers for the 0, 100, 300, 1200 and 2000 ppm groups were 98.8 (±3.4), 304.4 (±9.1), 1178.1  (±69.1) and 2028.2 (±107.3) ppm, respectively.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Read across from an analogous metabolite.
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1ff

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, table in chapter 1.2 and endpoint specific chapters
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
5-month-old
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
destilled water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solutions were verified to be within 10% of theoretical concentrations.
Details on mating procedure:
artificially inseminated
Duration of treatment / exposure:
gd 6–19
Frequency of treatment:
daily
Duration of test:
gd 30
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
No. of animals per sex per dose:
23-24
Control animals:
yes
Details on study design:
- Dose selection rationale not presented
Maternal examinations:
At scheduled necropsy on gd 30, maternal liver and kidneywere recorded. Kidneys were examined histologically in 10–17 dams/group.
At necropsy, 20–22 dams and litters/group were evaluated in the control and 3 lowest dose groups; 9 does and litters were evaluated in the highest dose group due to a high mortality rate.
Ovaries and uterine content:
At scheduled necropsy on gd 30, gravid uterus weights were recorded. Ovarian corpora lutea were counted and uterine implantation sites were recorded.
Fetal examinations:
All live fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and variations. Viscera were examined according to the Staples method and the skeleton was stained with Alcian Blue/Alizarin Red S. Heads from half the fetuses were fixed in Bouin’s solution and examined.
Statistics:
The litters were considered the experimental unit. Data were analyzed with the General Linear Trend Models procedures for ANOVA, Bartlett’s test for homogeneity of variance, Williams’ and Dunnett’s multiple comparison tests, and/or Fisher’s Exact Probability Test.
Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
At 2,000 mg/kg bw/day, 42% of the does died.
Body weight and weight changes:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Kidney weights were slightly increased in the 2,000 mg/kg bw/day group (not statistically significant).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy revealed renal toxicity including tubule dilatation and degeneration, epithelial necrosis, and intraluminal oxalate crystal deposition.
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
At 2,000 mg/kg bw/day, one doe aborted.
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): At 2,000 mg/kg bw/day, three does delivered early.
Other effects:
effects observed, treatment-related
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
gross pathology
mortality
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: no adverse effects observed
Developmental effects observed:
no
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1 076 mg/m³
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, 1,4-BDDMA does not need to be classified for toxicity to reproduction, developmental toxicity and teratogenicity 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.

Additional information