Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are no data available for 1,6-HDDMA itself.

For the evaluation of 1,6-HDDMA the data of the metabolite methacrylic acid and its donor substance methyl methacrylate, respectively, and 1,6 -Hexanediol are used. Read-across to the metabolites is justified by the fact that carboxylesterases are ubiquitous in the body and half-lifes of the other category substances are only a few minutes (see chapter 5.1 in the category document). In the body, methyl methacrylate hydrolyses rapidly to methacrylic acid and thus serves as methacrylic acid donor in several test systems investigating systemic effects.

MMA (methacrylic metabolite donor substance)

OECD 416 fertility, rat, gavage: parenteral NOAEL 400 mg/kg bw/d/ NOEL 50 mg/kg bw/d based on food consumption), offspring NOAEL 400 mg/kg bw/d (BASF 2009)

Summary

Based on the results of a reliable fertility study with the methacrylic metabolite donor substance MMA and the alcohol metabolite 1,6 -HD, exposure to 1,6-HDDMA is not likely to result in reproductive toxicity.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0 generation parental animals: 25
F1 generation parental animals: 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
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
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:
food consumption and compound intake
Dose descriptor:
LOEL
Remarks:
food consumption
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: consequence of reduced appetite observed in the F0 parental females
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects observed
Dose descriptor:
NOAEL
Remarks:
General systemic toxicity
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects observed
Critical effects observed:
no
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
Remarks:
food consumption
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
Remarks on result:
other:
Remarks:
effects on food consumption beeing a consequence of reduced appetite observed at the LOEL of 150 mg/kg bw/d in the F0 parental females
Dose descriptor:
NOAEL
Remarks:
fertility & reproductive performance
Generation:
F1
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects observed
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no dverse effects observed
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

There are no data available for 1,6-HDDMA itself.

For the evaluation of 1,6-HDDMA the data of the metabolite methacrylic acid and its donor substance methyl methacrylate, respectively, are used. Read-across to the metabolites is justified by the fact that carboxylesterases are ubiquitous in the body and half-lifes of the other category substances are only a few minutes (see 5.1). In the body, methyl methacrylate hydrolyses rapidly to methacrylic acid and thus serves as methacrylic acid donor in several test systems investigating systemic effects.

MMA (methacrylic metabolite donor substance)

Methyl methacrylate (MMA) is the methyl ester of methacrylic acid (MMA) and is rapidly absorbed and metabolised to MAA within the body. It therefore acts as an effective systemic deliver system for MAA avoiding the local high toxicity of MMA due to its acidity. The reference chemical for the methacrylic moiety of the category members, MMA, has been tested in an OECD TG 416 oral two-generation reproduction toxicity study in rats, in which both, parental and F1 animals were dosed with 0; 50; 150 and 400 mg/kg bw/day (BASF, 2009).

In mid- and high-dose parental animals (150 and 400 mg/kg bw/d) temporary salivation, presumably due to a bad taste of the test substance and associated dose-related intermittent reductions of food consumption were noted. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group and not associated with effects on histopathology or reproductive performance.

The NOAEL for fertility and reproductive performance for the P and F1 parental rats was determined to be 400 mg/kg bw/day, the highest dose tested. The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance was determined to be 400 mg/kg bw/day, the highest dose tested. There were no signs of systemic toxicity other than reduced body weight gain associated with reduced food consumption, presumably due to bad palatability (NOEL 50 mg/kg bw/day for the P and F1 parental rats).

The 2-generation study with MMA provides confidence that the absence of reproductive effects below maternal toxic doses seen at in the screening studies with the three category members, are a reliable indication for an absence of fertility effects below maternal toxic doses in higher tier studies such as a 2 generation study when considering the methacrylic moiety of the category members.

1,6 -HD (alcohol metabolite of 1,6-HDDMA)

A screening study according to OECD 421 in Wistar rats with 1,6-HD was performed in which animals were tested at gavage doses of 0, 100, 400, 1000 mg 1,6-HD /kg/day. The body weight gain of high dosed F0 males was retarded as sole effect in this study. There were no signs for impairment of reproductive function of F0 rats and no signs of developmental toxicity in their offspring. Therefore, the NOAEL for parental toxicity is 1000 mg/kg/d for females and 400 mg/kg bw for males; the NOAEL for reproductive function and for developmental toxicity is 1000 mg/kg/d (OECD 2002).

 

Compliance to REACh requirements

The screening study requirement is considered as covered with a reliable OECD 416 oral rat study, performed with the metabolite donor substance MMA, plus an OECD screening study with the alcohol metabolite 1,6-HD in rats. The read across is done with a high level of confidence (see chapter “Toxicokinetics” and the attached category document).

Effects on developmental toxicity

Description of key information

There are no data available for 1,6-HDDMA itself.

For the evaluation of 1,6-HDDMA the data of the metabolite methacrylic acid and its donor substance methyl methacrylate, respectively, are used. Read-across to the metabolites is justified by the fact that carboxylesterases are ubiquitous in the body and half-lifes of the other category substances are only a few minutes (see 5.1). In the body, methyl methacrylate hydrolyses rapidly to methacrylic acid and thus serves as methacrylic acid donor in several test systems investigating systemic effects.

MMA (methacrylic metabolite donor substance)

OECD 414 developmental, rabbit, gavage: parenteral NOAEL 50 mg/kg bw/d, developmental NOAEL 405 mg/kg bw/d (BASF 2009; study selected from a larger data set for REACh requirement reasons)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
Himalayan
Details on test animals 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
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
actual dose: 41 mg/kg bw/d
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
actual dose: 132 mg/kg bw/d
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
actual dose: 406 mg/kg bw/d
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:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
other: no adverse effects observed; actual dose 406 mg/kg/d
Dose descriptor:
NOEL
Remarks:
food consumption
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
Remarks on result:
other: actual dose: 41 mg/kg/d
Abnormalities:
effects observed, treatment-related
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
Remarks:
prenatal developmental toxicity
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no

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 450 mg/kg bw/d and the no observed effect level (NOEL) for maternal toxicity is 50 mg/kg bw/d based on effects on food consumption. 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 nominal 450 mg/kg bw/d (actual 406 mg/kg bw/d), the highest dose tested. The no observed effect level (NOEL) for maternal toxicity is nominal 50 mg/kg bw/d (effective 41 mg/kg bw/d) 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 (actual 132 mg/kg bw/d).

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is nominal 450 mg/kg bw/d (actual 406 mg/kg bw/d). No adverse fetal findings of toxicological relevance were evident at any dose.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CDBR
Details on test animals 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)
Dose / conc.:
99 ppm
Remarks:
corresponding to 412 mg/m3 or 0.41 mg/L
Dose / conc.:
304 ppm
Remarks:
corresponding to1285 mg/m3 or 1.29 mg/L
Dose / conc.:
1 178 ppm
Remarks:
corresponding to 4900 mg/m3 or 4.9 mg/L
Dose / conc.:
2 028 ppm
Remarks:
corresponding to 8436 mg/m3 or 8.44 mg/L
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
Remarks:
maternal toxicity
Effect level:
ca. 0.41 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Remarks:
maternal toxicity
Effect level:
8.44 mg/L air (analytical)
Based on:
test mat.
Remarks on result:
other: no adverse effects observed
Abnormalities:
no effects observed
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
Remarks:
fetotoxicity
Effect level:
8.44 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: corresponding to 2028 ppm; no substance related effects observed
Dose descriptor:
NOAEC
Remarks:
teratogenicity
Effect level:
8.44 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: corresponding to 2028 ppm; no substance related effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
8 436 mg/m³
Study duration:
subacute
Species:
rat
Additional information

There are no data available for 1,6-HDDMA itself.

For the evaluation of 1,6-HDDMA the data of the metabolite methacrylic acid and its donor substance methyl methacrylate, respectively, are used. Read-across to the metabolites is justified by the fact that carboxylesterases are ubiquitous in the body and half-lifes of the other category substances are only a few minutes (see 5.1). In the body, methyl methacrylate hydrolyses rapidly to methacrylic acid and thus serves as methacrylic acid donor in several test systems investigating systemic effects.

MMA (methacrylic metabolite donor substance)

In a developmental toxicity study according to OECD 414 MMA (methyl methacrylate) was administered by inhalation exposure to 99, 304, 1178, and 2028 ppm (412, 1285, 4900, 8436 mg/m³; Solomon et al., 1993). No relevant maternal, treatment-related effects, except reduced body weight, were noted at any concentration tested. No embryo or 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 was 450 mg/kg bw/d. No adverse foetal findings of toxicological relevance were evident at any dose, even in the presence of maternal toxicity (BASF, 2009). MMA is not a selective teratogen.

 

Summary

Based on the results of reliable developmental toxicity studies with the alcohol metabolite EG and the methacrylic metabolite donor substance MMA, exposure to 1,6HDDMA is not likely to result in developmental toxicity.

Justification for classification or non-classification

In the absence of toxicity to reproduction (fertility and developmental effects) of its metabolites MAA and 1,6 -HD, no classification is required.