N-[3-(dimethylamino)propyl]methacrylamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

An extended one-generation reproductive toxicity study (OECD 443) in rats via the oral route was performed. Due to several compliance and quality issues, there is an ongoing discussion with ECHA on the reliability of the study. 

Screening (OECD 422)

NOAEL oral/rat; parenteral toxicity: 200 mg/kg bw/d; reproduction toxicity 400 mg/kg bw/d according to OECD 422 (RTC 2002; valid without restrictions, reliability 1, with GLP).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined repeated dose and reproduction / developmental screening study in rats

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb, 2001 to 16 Oct, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 422, Screening test, GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted on 22 March 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): N-3-Dimethylaminopropyl methacrylamide
- Supplier: Evonik Röhm GmbH, D-64293 Darmstadt, Germany
- Substance type: organic
- Physical state at room temperature: liquid
- Stability under test conditions: Stability in water: > 160 hours in water; pure: stable for 3 month
- Storage condition of test material: 4 °C, light protected

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hartlan Italy S.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: (P) Males/females: approximately 12 wks
- Weight at study initiation: (P) Males: 270 - 298 g; Females: 195 - 214 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 (Altromin MT Altromin, D-32770 Lage, Postfach 1120, Germany)
- Water: ad libitum, supplied via water bottles
- Acclimation period: 25 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:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: A weighted amount of the test item was dissolved in the vehicle (distilled water) and brought to the final
volume appropriate for each concentration. The test solutions were prepared daily at room temperature (concentrations of 5, 10, 20 and
40 mg/ml). All test item concentrations and dosages were based on the test item as supplied (dose volume of 10 ml/kg body weight).
Dose volumes for males were calculated according to individual body weight on the first day of treatment and adjusted according to individual
body weight at weekly intervals thereafter. Dose volumes for females were calculated according to individual body weight on the first day of
treatment and adjusted according to individual body weight at weekly intervals up to positive identification of mating. Dose volumes were adjustced to body weight on Days 0, 7, 14 and 20 post-coitum and on Day 0 post-partum. Thereafter individual dose volumes remained constant.
Control animals received the vehicle alone at the same dose volume.

Details on mating procedure:

- M/F ratio per cage: Mating was monogamous (one male to one female).
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: The presence of a vaginal plug or sperm in the vaginal smear was taken as positive identification of mating, upon which vaginal smearing ceased.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Once during week 1 and again during the last week of treatment, samples of prepared formulations were analysed for verification of concentration.

Duration of treatment / exposure:

Males
Treatment commenced when the males were approximately 13 weeks old. It continued for two weeks prior to pairing and through the mating period.
The males were killed after at least 4 weeks of treatment.
Females
Treatment commenced when the females were approximately 13 weeks old. It continued for two weeks prior to pairing, through the mating and
gestation periods up to day 3 of post-partum. Dams and offspring were sacrificed on day 4 post-partum.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0 (only vehicle), 50, 100, 200 and 400 mg/kg/day
Basis:
nominal in water

No. of animals per sex per dose:

10 male and 10 female per dose group

Control animals:

yes

Details on study design:

- Dose selection rationale: The oral route was selected as it is a possible route of exposure of the test item in man. The dose levels of 50, 100, 200
and 400 mg/kg/day were defined on the basis of the 14 day oral toxicity range finding study (see chapter 7.5.1, Evonik Röhm GmbH, 2001).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
All clinical signs were recorded for individual animals. Examination of individual animals for signs of reaction to treatment was carried out daily
before dosing, immediately after, and approximately 30 minutes and 1 hour after dosing.
Animals were subjected to a detailed clinical examination at weekly intervals. For male animals the clinical signs were performed before treatment
started until necropsy. For female animals clinical signs were performed before treatment started, weekly during the treatment and the mating
periods and on days 0,7, 14 and 20 of gestation and on day 2 post-partum.
Animals were examined in an open arena for a period of three minutes.


BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed at allocation (Study Day -14), at treatment initiation
(Study Day I), and then weekly to pairing. Males were then weighed at weekly intervals up to the day sacrifice, females were weighed on Days 0, 7, 14
and 20 post-coitum and 0/1 and 4 post-partum.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: The weight of food consumed by each cage of rats was recorded weekly following allocation to pairing. Food consumption for
each female was calculated on gestation Days 7, 14 and 20 and on post-partum day 4.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: The total litter size (live and dead) was counted alter parturition (Day 0). Live
pups were identified individually within the litter by toe amputation, sexed, and examined for external abnormalities. All pups were examined daily for morbidity and abnormalities dead or abnormal young. All pups were individually weighed on post-partum days 0 and 4. The sexing at birth was
rechecked on Day 4 post-partum.

Postmortem examinations (parental animals):

SACRIFICE
All adult animals were killed with carbon dioxide at the end of the scheduled treatment period (day 31 and day 32 of the study for males and on
post partum day 4 for females). Nonpregnant females were killed after Day 25 post-coitum. The number of visible implantation sites were recorded
for each dam. Uteri of nonpregnant females were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantatlon.
All pups were killed by intracapular injection of "Tanax".

GROSS NECROPSY
- Gross necropsy: The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surfaces and orifices). Changes were noted and the requisite organs weighed.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs from all animals in all groups were dissected free of fat and weighed:
Testes, Epididymides, Ovaries with oviduct, Uterus with cervix, Prostate and seminal vescicles

In addition from at least 5 male and female animals per group the following organs were dissected free of fat and weighed:
Adrenals, Brain, Heart, Kidneys, Liver, Thymus, Spleen
This was a deviation from the protocol which indicated 5 animals per sex only.

Tissues fixed and preserved
From at least 5 male and female animals per group the following tissues listed below were fixed and preserved in 10% buffered formol-saline (except
testes and epididymides which were fixed in Bouin's solution and preserved in 70% ethyl alcohol).
Abnormalities Lymph nodes - mesenteric
Adrenal glands Ovaries with oviduct
Brain (only for females) Prostate
Caecum Rectum
Colon Sciatic nerve
Duodenum Seminal vescivles
Heart Spleen
Ileum Stomach
Jejunum Testes
*Kidneys Thymus (where present)
*Liver Thyroid gland
Lungs Trachea
Lymph nodes - cervical Urinary bladder
Uterus with cervix
This was a deviation from the protocol which indicated only 5 males and 5 females.

Histopathological examination
After dehydration and embedding in paraffin wax, sections of the tissues listed above were cut at 5 micrometre thickness and stained with
haematoxylin and eosin. Additional sections were stained with periodic acid Schiff s (PAS). The PAS stained sections were used for testes and
epididymides to identify spermatogenic stages. In the first instance the examination was restricted as detailed below:
Tissues specified above from all animals selected from the control and the high-dose group. As treatment related changes were observed jn high-
dose animals, when compared with controls, the evaluation was then extended to the testes and epididymides of the low and intermediate dose
groups.

Statistics:

Standard deviations will be calculated as appropriate. For contiunous the significance of the differences amongst group means will be assessed by
analysis of variance. Differences between each treated group and the control group will be assessed by Dunnett's test using a pooled error
variance. The homogeneity of the data was verified by Bartlett's Test before Dunnett's Test was performed. If the data were found to be
inhomogeneous, a modified T Test (Cochran and Cox) was applied. The non-parametric Kruskal-Wallis analysis of variance was used for litter and sex ratios data. Intergoup differences between the control and treated groups were assessed by the non-parametric version of the Williams test.
The criterion for statistical sigificance was p<0.05. The mean values, standard deviations and statistical analysis were calculated from actual
values in the computer without rounding off.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No deaths occurred during the course of the study.
No significant daily post-dose observations were observed during the study. Only on one occasion, one high-dose male showed rales. These data
are not tabulated in this report.
Detailed clinical signs with neurotoxicity assessment did not show any signs which could be related to the treatment with the test item.
No toxicological importance was attributed to the small mass observed in the perigenital area in one male animal in group 5 and one control male
(not confirmed at necropsy) on the day of sacrifice.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight and body weight gain were unaffected by treatment.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Food consumption was comparable between groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No differences in fertility parameters were observed that could be related to treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
One male in the control group, one male in group 3 and one male in group 4 failed to induce pregnancy. One male and one female in the control
group did not mate after 14 days of cohabitation (maximum allowed). Mating was not detected for two females in group 2 and one female in group 5. One female in the control group, one female in group 3 and one female in group 4 proved not to be pregnant at necropsy. One female in group
4 had total litter loss on day 4 of lactation. The number of females with live pups at Day 4 post-partum was 8 in the control group, 10 in group 2, 9 in group 3, 8 in group 4 and 10 in group 5.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No differences between groups were observed in terminal body weight. The statistically significant increase in absolute and relative heart weight
observed in females of group 4 was not considered of toxicological significance. A statistically significant increase in absolute spleen weight was
observed in high-dose group females when compared to controls. A dose related increase, achieving statistical significance for the high-dose group, in relative liver and kidney weights was observed in male animals compared to controls. Statistically significant increases in relative spleen
weights, with a dose relationship, were observed for females of the two higher-dose groups investigated. These changes were considered related to
treatment.

MACROSCOPIC OBSERVATIONS (PARENTAL ANIMALS)
No change was observed in the treated animals which could be considered related to treatment.

MICROSCOPIC OBSERVATIONS (PARENTAL ANIMALS)
Unilateral, slight to moderate tubular atrophy was observed in the testes of 3/5 high dose group males, no control animal showing a similar lesion. In addition, a unilateral sperm granuloma and reduction of sperm were also described in the epididymides of two males, respectively.
No similar change was described in the testes and cpidldymides of the animals in groups 2,3 and 4. Increased incidence of slight to mild, foca/
multifocal chronic inflammation was reported in the lungs of group 5 animals, both sexes, when compared with the control group. This finding was
considered incidental in origin.
The remaining lesions were seen to be expression of spontaneous pathology normally detected in animals of this species and age, under our
experirnerltal conditions.

Spermatogenic staging
Identification of the stages of the spermatogenic cycle (as described by Leblond and Clermont, 1952) were performed in the animals of control and
high dose groups.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within
the different stages.
Testicular atrophy was observed in 3/5 high dose group animals. This change was considered to be reversible. The epididymidal tubules of these
animals contained numerous exfoliated degenerated germ cells with reduction or absence of sperm (oligospermia) and in some instances associated with interstitial fibrosis. Sperm granuloma was detected in one of these animals.
On the basis of these results obtained in the animals of the high dose when compared with controls, the identification of the stages of the
spermatogenic cycle were extended to the testes and epididymides of the remaining groups.
These animals did not show the histopathological changes detected in the animals of the high dose group. The only change detected was some
irregularly arranged seminiferous tubules, but with normal germinal epithelium in two animals in group 3 and in group 4.

HISTOPATHOLOGY (PARENTAL ANIMALS)
A slight, but statistically significant reduction in mean red blood cell volume and mean corpuscular haemoglobin and an increase in white blood cell
count was observed in males of the high-dose group. No toxicological significance is given to the slight statistically significant increase in red blood cell count observed in high-dose females. These slight differences, within the normal range for this species at thIs age, are not considered of
toxicological significance.


OTHER FINDINGS (PARENTAL ANIMALS)

Implantation and pre-birth loss data:
Gestation periods were similar in all groups. All dams gave birth withn day 22 post-coitum. Implantation and pre-birth loss were similar between the
control and the treated groups.

Motor activity and sensor reactivity to stimuli:
No signs that could be related to treatment were seen.

Clinical chemistry:
A slight, but statistically significant reduction in sodium and potassium was observed in females of group 3 and in males of group 5. respectively. In
addition, in males of group 4 a slight statistically significant increase in chloride was observed. No toxicological significance was attributable to the
statistically significant increase in total bilirubin observed for males of groups 2 and 5 since it was within the normal range for animals of this age and species.
A statistically significant increase in total cholesterol and total protein was observed in high-dose females compared to controls.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

IMPLANTATION and pre-birth loss data:
Gestation periods were similar in all groups. All dams gave birth within day 22 post-coitum.
Implantation and pre-birth loss were similar between the control and the treated groups.

VIABILITY (OFFSPRING)
F1 litter viability and growth and sex-ratios
Litter data and sex ratios were unaffected by treatment.

CLINICAL SIGNS (OFFSPRING)
Pre-weaning clinical signs did not show treatment related effects.

NECROPSY FINDINGS (OFFSPRING)

Necropsy findings in decedent pups did not show any abnormalities that could be related to treatment.
Necropsy findings in F1 pups at Day 4 post-partum did not show any treatment-related effects.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Reproductive effects observed:

not specified

Conclusions:

N-3-Dimethylaminopropylmethacrylamide was administered to male and female animals 2 weeks prior to pairing and throughout gestation and
lactation periods up to post-partum day 3 at dosages of 50, 100, 200 and 400 mg/kg/day.
No effects were observed up to the highest dose for the mean pre-coital intervals, the fertility index, litter data or the gestation length. No treatment related effects were observed on necropsy in decedent and F1 pups nor in the macroscopic examination of the parental generation.

A statistically significant increase in absolute and relative spleen weight was observed in high-dose females compared to controls. In addition, an
increase in relative spleen weight was noted in females receiving 200 mg/kg/day.
A dose related increase, achieving statistical significance for the high-dose group, in relative liver and kidney weights was observed in treated males when compared to controls. These changes were considered to be treatment related.
At microscopic observations, unilateral, slight to moderate tubular atrophy was observed in the testes of 3/5 high-dose group males. In addition,
unilateral sperm granuloma and reduction of sperm were also described in the epididymides of two males, respectively. On the basis of these
treatment related changes, the microscopic evaluation was then extended to testes and epididymides of the remaining dosages. No similar change
was described in the testes and epididymides of the animals of group 50 mg/kg/day, 100 mg/kg/day and 200 mg/kg/day. On the basis of these
results, NOAEL= 200 mg/kg/day.

Executive summary:

The toxicity of N-3-Dimethylaminopropylmethacrylamide (purity: 99.37%, CAS: 5205- 93-6) when given by daily oral administration to rats for two weeks before mating, during mating and until the day before sacrifice (at least 4 weeks of treatment for males and Day 3 post-partum for females) has been investigated. Four groups, each of 10 male and 10 female Sprague Dawley rats, received the test item by oral gavage at dosages of 50, 100, 200 and 400 mg/kg/day. A fifth similarly constituted group received the vehicle alone (distilled water) and acted as a control.

No signs were noted at post-dose observations. Detailed examination of clinical signs with neurotoxicity observations did not show any signs attributable to treatment.

Body weight and food consumption were unaffected by treatment.

Reproduction parameters were unaffected by treatment.

Litter data, implantation loss and gestation length were comparable between groups.

No toxicological significance was given to the slight statistically significant changes in some haematology or clinical chemistry parameters observed occasionally in treated animals when compared to controls.

A statistically significant increase in absolute and relative spleen weight was observed in high-dose females compared to controls. In addition, an increase in relalive spleen weight was noted in females receiving 200 mg/kg/day. A dose related increase, achieving statistically significance for the high-dose group, in relative liver and kidney weights was observed in treated males when compared to controls. These changes were considered to be treatment related.

No changes were observed in treated animals at macroscopic observations which could be considered related to treatment.

Unilateral, slight to moderate tubular atrophy was observed in the testes of 3/5 high-dose group males. This change was considered to be reversible. In addition, a unilateral sperm granuloma and reduction of sperm were also described in the epididymides of two males, respectively. On the basis of these treatment related changes, the microscopic evaluation was then extended to testes and epididymides of the remaining dosages. No similar change was described in the testes and epididymides of the animals of group 2,3 and 4.

On the basis of these results the NOAEL was considered to be 200 mg/kg bw/day in males and females.

This study is acceptable and satisfies the guideline requirement for a screening reproductive study (OECD 422) in rats.

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

Quality of whole database:

Screening level data available from OECD 422 study with high reliability (1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

N-3-Dimethylaminopropylmethacrylamide has been tested in an OECD 422 combined repeated dose toxicity study with reproduction/developmental toxicity in rats at oral doses of 0(water), 50, 100, 200 and 400 mg/kg/day. Signs of parental toxicity were seen at the highest dose (400 mg/kg bw/d).

Among the relevant findings at this dose was a statistically significant increase in absolute and relative spleen weight in high-dose females compared to controls. In addition, 3/5 male animals at the same dose showed slight testicular atrophy which was considered to be reversible and a statistically significant increase in relative liver and kidney weights which was considered to be related to treatment. The systemic NOAEL was reported as 200 mg/kg/d. Subsequently, an oral 90 day study was performed, which reported a systemic NOAEL of 300 mg/kg bw/d.

The reproductive parameters in the OECD 422 study were unchanged up to the highest dose of 400 mg/kg bw/d. The slight testicular atrophy observed in 3/5 investigated male rats at 400 mg/kg bw/d apparently had no influence on reproductive performance.

While the sight effects in a few male animals could inidcate reprotoxic and endocrinedisrupting potential, there is no indication in the most relevant 90 d study. Therefore, there are no such effects expected for this substance.

Therefore, the NOAEL of 300 mg/kg bw/d in the 90d study is regarded as the reproductive NOAEL for males while the reproductive NOAEL for effects in females and F1 was 400 mg/kg bw/d. It is concluded that specific reproductive toxicity of DMAPMA is absent (see also argumentation in endpoint summary for Repeated dose toxicity).

Also, for the two structural constituents of DMAPMA – methacrylic acid and dimethyminopropylamine, the prevalent reproductive toxicity studies are negative, indicating a general absence of structural alerts with regard to a specific reproductive toxicity of N-(3-Dimethylaminopropyl)-2- methacrylamide.

Short description of key information:
NOAEL oral/male/ reproduction: 300 mg/kg bw/d
NOAEL oral/female/reproduction toxicity: 400 mg/kg bw/d

Effects on developmental toxicity

Description of key information

NOAEL oral/rat/female; maternal & developmental toxicity: 400 mg/kg bw/d according to OECD 414 (RTC 2013; valid without restrictions, reliability 1, with GLP).

NOAEL oral/rabbit/female; maternal & developmental toxicity: 180 mg/kg bw/d according to OECD 414 (RTC 2018; valid without restrictions, reliability 1, with GLP).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Febuary 2013 - August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 414, GLP. Method and results sufficiently described.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): N-(3-Dimethylaminopropyl) methacrylamide
- Supplier: Evonik Röhm GmbH, D-64293 Darmstadt, Germany
- Substance type: organic
- Physical state at room temperature: liquid
- Stability under test conditions: Stability in water: > 160 hours in water; pure: stable for 3 month
- Storage condition of test material: room temperature, light protected

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: 9 weeks old (females), 11 weeks old (males)
- Weight at study initiation:females 206 to 230 g, males at least 350 g)
- Fasting period before study: no data
- Housing: during pre-pairing period and after mating animals were housed no more than 5 of one sex to a cage in polisulphone cages (59.5 x 38 x 18.5cm); during mating period, the rats were housed 1 male to 1 female in clear polycarbonate cages (42.5 x 26.6 x 18.5 cm)
- Diet (e.g. ad libitum): ad libitum (4 RF 21, Mucedola S.r.1., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2°C
- Relative humidity: 55 ± 15%
- Air changes (per hr): 15 to 20 cycles/hour
- Photoperiod (hrs dark / hrs light): artificial light 12 h/12 h

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): The required amount of N-(3 dimethylaminopropyl)methacrylamide was dissolved in the vehicle
(distilled water). The formulations were prepared daily (concentrations of 5, 15 and 40 mg/mL) 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:

Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (95-105%) with the exception of Group 2. Analysis was repeated on a new preparation and results were within the acceptability limits.
In this study a 24 hour stability at room temperature was verified in the range from 5 to 40 mg/mL.
According to RTC SOPs, solutions are considered to be stable if concentration after the defined period of storage is still acceptable (95-105%).

Details on mating procedure:

Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made. The presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day0 of gestation.

Duration of treatment / exposure:

6 - 19 day post coitum inclusive

Frequency of treatment:

once a day

Duration of test:

14 d (dams were euthanized on gestation day 20)

No. of animals per sex per dose:

24 mated female rats per group exposed.

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Animals were observed daily for behavioral changes.

DETAILED CLINICAL OBSERVATIONS: Yes (starting from allocation until sacrifice)
- Time schedule: once a day; Morbidity and mortality: at least twice a day including weekends and public holidays, once a day on other days

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were recorded on GD0, 3, 6, 9, 12, 15, 18 and 20 p.c.starting from Day0 post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption: The quantity of food consumed by each female was measured on Days 3, 6, 9, 12, 15, 18 and 20 p.c.starting from Day 0 post coitum. 

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20

Ovaries and uterine content:

The ovaries and uterus of the females were examined to determine:
gravid uterine weight (will not be obtained from animals found dead or killed during the study), number of corpora lutea, number of implantation sites, number, sex and weight of all live foetuses, number and sex of dead fetuses (foetuses at term without spontaneous movements and breathing), number of intra-uterine deaths and gross evaluation of placentae.
A gross evaluation of placentae was also undertaken.

Fetal examinations:

- External examinations: Yes: all live foetuses
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

Continuous data (for instance body weight, food consumption, …) amongst group means were assessed by Dunnett's test or a modified test, depending on the homogeneity of data.
Statistical analysis of non-continous data are carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse maternal findings of toxicological relevance were evident at any dose.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse fetal findings of toxicological relevance were evident at any dose.

Abnormalities:

not specified

Developmental effects observed:

not specified

Mortality and fate of females

 

No animals died during the study.

All females were found pregnant at necropsy.

The number of females with live foetuses on gestation Day 20 was 24 in each of the control, low, mid- and high dose groups.

 

Clinical signs

 

No signs of toxicological significance were noted during the study and no signs of reactions to treatment were observed during the dosing period.

 

Body weight and body weight gain

 

No differences in body weight were noted between control and treated groups.

At body weight gain, a statistically significant increase of approximately 21% was noted on Day 6 post coitum in females receiving 400 mg/kg/day and a statistically significant decrease of approximately 29 and 67% was noted on Day 9 post coitum in females receiving 150 and 400 mg/kg/day respectively, when compared to controls. These changes were considered of no toxicological relevance.

 

Food consumption

 

The statistically significant decrease of approximately 11%, detected on Day 9 post coitum in females receiving 400 mg/kg/day, was not considered of toxicological significance.

 

Terminal body weight, uterus weight and absolute weight gain

 

No significant differences in terminal body weight, gravid uterus weight and absolute weight gain were observed in treated groups, when compared to the control group.

Litter data and sex ratios

 

Litter data, mean foetal weight and sex ratios were not affected by treatment.

 

Macroscopic examination of females

                                                                

No treatment-related changes were observed at post mortem examination in treated animals, when compared to the controls.

 

External examination of foetuses

 

The malformation agnatia was detected in one foetus of the mid-dose group, one dead foetus was detected in the high dose group and small foetuses were detected in control and treated groups.

A total of 10 small foetuses (< 2.7 g) were detected; six out of 349 in control females, one out of 367 in low dose females, one out of 344 in mid-dose females and two out of 348 in high dose females.

All these findings were considered incidental and not dose-related.

 

Skeletal examination of foetuses

 

Malformations were detected in control, low and mid-dose groups: no ossification of pubis in one foetus of the control group (associated with no ossification of ischium), one foetus of the low dose group and two foetuses of the mid-dose group. In addition in the mid-dose group malformation such as absence of one rib and absence of mandible was also detected in two foetuses of two different litters.

Considering that some of these alterations were observed in small foetuses (foetal weight < 2.7 g) and were not dose-related, they were considered incidental.

 

Visceral examination of foetuses

 

No findings that could be considered treatment-related were noted at visceral examination of the foetuses in any group.

Conclusions:

In a developmental toxicity study according to OECD 414 N-Dimethylaminopropyl methacrylamidewas administered to female rats dosed dissolved in distilled water by gavage at dose levels of 0, 50, 150 and 400 mg/kg bw/day from days 6 through 19 of gestation. The maternal NOAEL in this study was found to be 400 mg/kg bw/day and the developmental NOAEL was also found to be 400 mg/kg bw/day.

Executive summary:

In a developmental toxicity study according to OECD 414 N-Dimethylaminopropyl methacrylamide was administered to female rats (Sprague-Dawley, (SD)) by oral gavage at dose levels of 0, 50, 150 and 400 mg/kg bw/day from days 6 through 19 of gestation.

 

The results showed that no animals died during the study. The number of females with live foetuses on gestation Day 20 was 24 in each of the control, low, mid- and high dose groups. No clinical signs of toxicological significance were noted during the study and no signs of reactions to treatment were observed during the dosing period. No differences of toxicological relevance were noted in body weight of females during the study, between control and treated groups. At body weight gain, a statistically significant increase of approximately 21% was noted on Day 6 post coitum in females receiving 400 mg/kg/day and a statistically significant decrease of approximatelly 29 and

67 % was noted on Day 9 post coitum in females receiving 150 and 400 mg/kg/day respectively, when compared to controls. These changes were considered of no toxicological relevance.

No relevant changes were detected in food consumption between treated and control females. No significant differences in terminal body weight, gravid uterus weight and absolute weight gain were observed in treated groups, when compared to the control group.

 

Litter data, mean foetal weight and sex ratios were not affected by treatment. No treatment-related changes were observed at post mortem examination in treated animals, when compared to the controls.

Agnatia was detected in one foetus of the mid-dose group, one dead foetus was detected in the high dose group and small foetuses were detected in control and treated groups. All these findings were considered incidental and not dose-related.

 

Malformations were detected in control, low and mid-dose groups. Considering that some of these alterations were observed in small foetuses (foetal weight < 2.7 g) and were not dose-related, they were considered incidental.

 

No findings that could be considered treatment-related were noted at visceral examination of the foetuses in any group.

 

The maternal NOAEL was therefore 400 mg/kg bw/day.

The developmental NOAEL was therefore 400 mg/kg bw/day.

 

None of the litter parameters recorded (implantations, live fetuses, percentage of male/female fetuses, fetal body weight) were affected.

There were no treatment-related malformations and there were no treatment-related variations that were considered to be adverse. 

 

N- Dimethylaminopropyl methacrylamide folmulation analyses were 95% to 105% of the nominal concentration and were within the acceptable range.

 

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rats.

 

 

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.

Reference 2

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

Remarks:

GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia S.r.l.
- Age at study initiation: females 13 to 18 weeks (males at least 25 weeks)
- Weight at study initiation: at least 2.5 kg
- Housing: The animals ( 2 females/cage at arrival, singly housed formDay 0 of gestation) were housed in polycarbonate/stainless steel cages with perforated NorylTM floor suspended over trays.
- Diet (e.g. ad libitum): Mucedola 2 RB 15, ad libitum
- Water (e.g. ad libitum): Drinking water ad libitum
- Acclimation period: at least 25 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The required amount of N-(3-DIMETHYLAMINOPROPYL) METHACRYLAMIDE was dissolved in the vehicle. The preparation were made daily (concentrations of 2, 6 and 18 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
VEHICLE
- Amount of vehicle (if gavage): dose volume of 10 mL/kg body weight. Dose volumes were calculated according to the most recently recorded body weight.

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Females were introduced to sexually mature males obtained from the same supplier. Each female remained with the male (in the home cage of the male) for at least 1 hour after successful mating was observed. Each female received 50 I.U. of luteinizing hormone in a marginal ear vein upon completion of the mating procedure. The day successful mating was detected was considered Day 0 post coitum (or gestation Day 0).

Duration of treatment / exposure:

All females were treated once a day from Day 6 through Day 28 post coitum.

Frequency of treatment:

once a day

Duration of test:

Treatment from Day 6 through Day 28 post coitum. Termination on Day 29 post coitum.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Group 1

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Dose / conc.:

180 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels of 20, 60 and 180 mg/kg/day were selected in consultation with the Sponsor based on information from a preliminary non GLP-compliant study.
In this study, the toxicity of N-(3-Dimethylaminopropyl) methacrylamide was investigated in non pregnant New Zealand White rabbits after daily oral administration for maximum 23 days in two groups with dosages of 200 mg/ kg bw/day and 400 mg/ kg bw/day.
Reduced faeces was noted as clinical sign at 400 mg/kg bw/d. A moderate reduction in bodyweight was noted in females treated at 400mg/kg/day starting from Day 4 of treatment. On Day 13 of treatment, a negative absolute weight gain was noted
in all females. The test item was considered not tolerated at this dose level and therefore the animals were sacrificed on Day 14 of the study. After 23 days of treatment with 200 mg/kg bw/d, a positive absolute weight gain was noted in 1/4 female, while
2/4 females did not have any increase, and 1/4 had a negative absolute gain of 100 g. No macroscopic observations were made in both treatment groups.
On the basis of the above mentioned results, the dosage of 400 mg/kg/day was considered not tolerated in rabbits. The dosage of 200 mg/kg/day is tolerated in non pregnant female rabbits. However, considering that the main study will comprise pregnant females and these animals are more sensitive than non pregnant ones, it was advisable to use a slightly lower dosage of 150 or 180 mg/kg/day as the high dose.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
– number and sex of dead foetuses (foetuses at termwithout spontaneous movements
and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae;
– individual placental weight.

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [all per litter ]
- Head examinations: Yes: [half per litter ]

Statistics:

Statistical analysis of litter data, sex ratio, uterus weight, absolute weight gain and corrected body weight non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a nonparametric version of theWilliams test. The significance of the differences amongst group means for the remaining data was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.

Indices:

Pre-implantation loss
Post-implantation loss
sex ratio
number of foetuses affected with structural deviations and the corresponding litter percentage

Clinical signs:

no effects observed

Description (incidence and severity):

Isolated cases of hairloss, staining or damaged ear observed both in controls and/or treated females are not considered related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality related to treatment occurred.

A total of two unforeseen deaths, one female in the low dose group and one female in the the high dose group occurred in the study. The low dose female no. 53 was found dead on Day 26 post coitum. The animal was not pregnant. Female no. 199 of the high dose group was sacrificed for an accidental trauma on Day 16 post coitum. The animal was pregnant. Both deaths were considered not treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant differences in body weight were seen.

A statistically significant reduction in body weight gain was observed in high dose females receiving 180 mg/kg/day on Days 9 and 12 of gestation (slighly negative body weight gain at both 3 day period vs. positive weight gain of 12-16 g per 3 day period in the control). Pregnant females of the mid-dose group receiving 60 mg/kg/day showed a reduction in absolute body weight gain in a single occasion, on gestation Day 9 (7 g vs 16 g in the control in this 3 day period). No effects were seen in low dose females receiving 20 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduction in food consumption of up to 25% compared to the control was observed in the high dose females compared to controls from Day 9 to 21 of gestation. A recovery was observed from Day 24 of gestation.

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 examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A reduction in uterus weight was noted in all treated females copared to controls but without a dose relation. This reduction was attributable to the lownumber of implantation observed the treated groups. The differences in number of implantation are not treatment related since animals were not treated during the implantation period (Days 0-5 of gestation).

No other organ weights examined.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The findings observed in the two unscheduled dead rabbits were considered incidental and therefore not treatment-related.
Animals killed at termination did not show relevant macroscopic changes. Agenesia of uterus (left horn) noted in one low dose female was consideres spontaneous in origin.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The reduction in the number of live foetuses observed in the high dose group is linked to the decrease in implants that occurred before the start of treatment.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Reduction in corpora lutea and the number of implantations
The formation of corpus luteum in the rabbit starts immediately after fertilisation at Day 0 of gestation and was not influenced by the treatment that began on Day 6 of gestation. Moreover, all data were within the range of historical control data of the supplier. Therefore, the diminution of the corpora lutea noted in the treated groups must be considered an incidental finding and consequently the decrease in the number of implants observed in the treated groups is considered not related to treatment.

Reduction in number of live foetuses and litter weight
The reduction in the number of live foetuses observed in the high dose group is linked to the decrease in implants that occurred before the start of treatment. Also here, the data were within the range of historical control data of the supplier. Mean foetal weight was comparable between groups, the reduction in litter weight observed in the high dose group is related to/ a consequence of the low number of live foetuses.

Dose descriptor:

NOAEL

Effect level:

180 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean foetal weight
was comparable between groups, the reduction in mean foetal weight observed in the high
dose group is related to the low number of live foetuses.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The reduction in the number of live foetuses observed in the high dose group is linked to the decrease in implants that occurred before the start of treatment.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No treatment-related findings were described.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal examination did not show any treatment related findings.

The degree of skeletal ossification observed in the treated groups was similar to the control group foetuses. The bifurcation of the 6th sternebra at the distal part observed in one foetus of the low dose group and one foetus of the mid-dose group is considered incidental. The observation was classified as malformation since it is a rare event, but considering the high incidence of asymmetrical ossification of bipartite sternebrae observed in control foetuses (up to 25% of litters affected), it is considered spontaneous in origin.

Visceral malformations:

no effects observed

Description (incidence and severity):

No treatment-related findings were described.

Examination of foetuses following freehand serial sectioning revealed a slight enlargement of the lateral brain ventricles (in few occasions the 3rd ventricle was also involved) in all groups. Although slight intergroup differences were recorded in the incidence, there was no consistent or dose-related association trend. In the control group, an increased incidence in moderate enlargements of brain ventricles was noted compared to the treated animals, demonstrating that this finding is commonly observed in control animals and confirming that the observations were not related to treatment. Retinal folds (slight) were observed in This is a commonly observed finding in rabbits and there is a strong evidence that slight retinal folds are artifactual in origin, and occur as a result of fixation in Bouin’s fluid. (Ref. Retinal folding in the termrabbit fetus—Developmental abnormality or fixation artifact?-. Reproductive Toxicology 26 (2008) 262–266).

Dose descriptor:

NOAEL

Effect level:

180 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Maternal toxicity

Signs of toxicity was present in the high dose animals receiving 180 mg/kg/day, demonstrated by a reduction of body weight gain on gestation Day 9 and 12. In addition, food

consumption was reduced from Day 12 to Day 21 of gestation. These treatment related effects did not influence the pregnancy status of the animals, no animals aborted or were sacrificed during the study. Recovery from the observed effects started to be evident on Day 15 of gestation for body weight and from Day 24 of gestation for food consumption. As animals recovered in the later study phase, these effects were not considered as adverse. The uterine examinations did not reveal any effects on foetuses in term of early or late resorptions and foetal death. At necropsy all animals at termdid not show abnormalities that could be considered related to treatment. On the basis of this results the dosage of 180 mg/kg/day is considered the NOAEL for dams.

Table 1: Overview on relevant key parameters

Group	Control (historical control data)	20 mg/kg bw/d	60 mg/kg bw/d	180 mg/kg bw/d
Body weight (kg) d 0 post coitum	3.62	3.55	3.60	3.57
Body weight gain (g) d 6 post coitum	21.5	18.2	21.7	18.5
Body weight gain (g) d 9 post coitum	16.0	12.5	6.5*C	-3.3 +C
Body weight gain (g) d 12 post coitum	12.1	9.4	14.5	-2.4 +D
Body weight gain (g) d 15 post coitum	19.0	25.7	25.9	4.7
Body weight gain (g) d 18 post coitum	5.8	7.5	-8.8	3.2
Body weight gain (g) d 21 post coitum	-5.6	-0.4	14.4	5.7
Body weight gain (g) d 24 post coitum	15.4	5.8	9.4	13.6
Body weight gain (g) d 27 post coitum	20.0	13.0	9.8	12.9
Body weight gain (g) d 29 post coitum	19.0	21.5	20.9	5.8
Gravid uterus weight (g)	568.8	489.8*KW	503.3*KW	454.2*KW
Corrected body weight (g)	3488	3432	3465	3363
Absolute weight gain (g)	-283	-231	-262	-341

*C = Cochran and Cox Test Significant at the 0.05 level

+C = Cochran and Cox Test Significant at the 0.01 level

+D = Dunnett LSD Test Significant at the 0.01 level

*KW = mean value of group is significantly different from control; Statistical analysis: Kruskall Wallis test; William’s test if group differences are different from control at p < 0.05

Developmental toxicity

The mean foetal weight of all treated groups was comparable to controls. No differences in sex ratios were observed. At the external and internal examination of foetuses, no treatment related abnormalities were described. Skeletal and fixed head examination did not revealed any treatment related findings. On the basis of these results the dosage of 180 mg/kg/day is considered the NOAEL for foetuses.

Table 2: Overview on key parameters

Group	Control (historical control data)	20 mg/kg bw/d	60 mg/kg bw/d	180 mg/kg bw/d
No. females mated and assigned to group (gestation phase)	25	25	25	25
Non-pregnant females	1	2	3	0
Humane kill - Accidental trauma	0	0	0	1
No. of females with abortions, early deliveries and stillbirths	0	0	0	0
No. of females at term (gestation Day 29) with live foetuses	24	23	22	24
Corpora lutea	10.52 (8.8-11.0)	9.61	9.23*#	8.92*#
Implantations	9.78	9.48	8.95	8.63
Pre-Implantation Loss total	0.74	0.13*	0.27	0.29
Pre-Implantation Loss %	7.22	1.35	2.70	3.38
Early Resorptions	0.09	0.26	0.27	0.42
Late Resorptions	0.22	0.26	0.18	0.33
Total Resorptions	0.30	0.52	0.45	0.75
Male %	50.74	45.78	45.32	46.88
Dead foetuses	0	0	0	0
Live foetuses	9.48 (7.0 -9.8)	8.96	8.50	7.88*#
Post-Implantation Loss total	0.30	0.52	0.45	0.75
Post-Implantation Loss %	3.08	6.61	5.77	7.48
Litter weight (g)	387.46	346.0	347.8	314.0*
Mean foetal weight (g)	41.38	38.88	41.62	40.56
Male  foetal weight (g)	41.63	39.22	41.54	40.54
Female  foetal weight (g)	40.53	38.38	41.53	40.10
Runts (foetuses with a weight <35 g) total litter/ total foetuses (% litter/ % foetuses)	11/39 (46/18)	13/47 (57/23)	10/27 (54/14)	13/28 (54/15)
Examination findings at necropsy: forelimbs with a bent shape	1	1	0	0
Foetal Fixed Head Examination - Brain: moderately enlarged ventricle  % litter/ % foetuses	17/8	4/1	5/1	0/0
Foetal Fixed Head Examination - Brain: slightly enlarged ventricle  % litter/ % foetuses	13/4	22/7	41/11	33/11
Foetal Fixed Head Examination - Eyes: folded retina  % litter/ % foetuses	17/6	35/12	27/8	17/8

* = mean value of group is significantly different from control; Statistical analysis: Kruskall Wallis test; William’s test if group differences are different from control at p < 0.05

# = within the range of historical control data of the supplier; referring to 36 full studies

Executive summary:

The effects of N-(3-DIMETHYLAMINOPROPYL) METHACRYLAMIDE on pregnancy and on embryo-foetal development were investigated in the rabbit, following oral administration (GLP study according to OECD 414). In-house mated females were randomly distributed to 4 groups with 25 females per group: 0/ 20/ 60/ 180 mg/kg bw/ day.

The test item was dissolved suspended in the vehicle (softened water) and administered from Day 6 through Day 28 post coitum at the dose volume of 10 mL/kg. Control animals

(Group 1) received the vehicle alone during the same treatment period, at the same dose volume. Body weight, daily clinical signs and food consumption were recorded during the

in vivo phase. All females were caesarean sectioned on Day 29 post coitum and subjected to post mortem examination. The number of corpora lutea, implantations, early and late

intra-uterine deaths, live foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external and internal abnormalities. Fixed-soft head from approximately half of the foetuses (i.e. routinely, every second live foetus) and skeletal examinations were performed in all groups.

Results

No mortality related to treatment occurred.

The number of females with live foetuses on gestation Day 29 was: 24 each in the control and high dose groups, 23 in the low dose group and 22 in the mid-dose group.

Isolated cases of hairloss, staining or damaged ear observed both in controls and/or treated females are not considered related to treatment.

No significant differences in body weight were seen. A statistically significant reduction in body weight gain was observed in high dose females receiving 180 mg/kg/day on Days

9 and 12 of gestation. Pregnant females of the mid-dose group receiving 60 mg/kg/day showed a reduction in absolute body weight gain in a single occasion, on gestation Day 9.

No effects were seen in low dose females receiving 20 mg/kg/day.

A statistically significant reduction in food consumption was observed in the high dose females compared to controls from Day 9 to 21 of gestation. A recovery was observed from

Day 24 of gestation.

A reduction in uterusweight was noted in all treated females copared to controls but without a dose relation. This reduction was attributable to the lownumber of implantation observed the treated groups. The differences in number of implantation are not treatment related since animals were not treated during the implantation period (Days 0-5 of gestation).

The reduction in the number of live foetuses observed in the high dose group is linked to the decrease in implants that occurred before the start of treatment. Mean foetal weight

was comparable between groups, the reduction in mean foetal weight observed in the high dose group is related to the low number of live foetuses.

No differences in sex ratios were seen between the control and the treated groups.

Macroscopic examination of dams: The findings observed in the two unscheduled dead rabbits were considered incidental and therefore not treatment-related. Animals killed at termination did not show relevant macroscopic changes. Agenesia of uterus (left horn) noted in one low dose female was consideres spontaneous in origin.

External and internal examinations of foetuses at necropsy: No treatment-related findings were described.

Foetal fixed head examination: No treatment related alteration in foetuses were described.

Skeletal examination did not show any treatment related findings.

Conclusion

Maternal toxicity

Signs of toxicity was present in the high dose animals receiving 180 mg/kg/day, demonstrated by a reduction of body weight gain on gestation Day 9 and 12. In addition, food

consumption was reduced from Day 12 to Day 21 of gestation. These treatment related effects did not influence the pregnancy status of the animals, no animals aborted or were

sacrificed during the study. Recovery from the observed effects started to be evident on Day 15 of gestation for body weight and from Day 24 of gestation for food consumption. As animals recovered in the later study phase, these effects were not considered as adverse. The uterine examinations did not reveal any effects on foetuses in term of early or late

resorptions and foetal death. At necropsy all animals at termdid not show abnormalities that could be considered related to treatment. On the basis of this results the dosage of 180

mg/kg/day is considered the NOAEL for dams.

Developmental toxicity

The mean foetal weight of all treated groups was comparable to controls. No differences in sex ratioswere observed. At the external and internal examination of foetuses, no treatment related abnormalities were described. Skeletal and fixed head examination did not revealed any treatment related findings. On the basis of these results the dosage of 180 mg/kg/day is considered the NOAEL for foetuses.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

180 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

All available studies are valid without restrictions, reliabilty 1 and performed with GLP, and cover two species (rat and rabbit).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There are three studies dealing with developmental toxicity available. All studies conducted by oral route are valid without restrictions, reliabilty 1 and performed with GLP. Based on the previous repeated dose and reproductive toxicity studies it is expected that the oral route is the best route of exposure providing adequate bioavailability of the test material.

In a reliable developmental toxicity study according to GLP and OECD 414, N-Dimethylaminopropyl methacrylamide was administered to female rats (Sprague-Dawley, (SD)) by oral gavage at dose levels of 0, 50, 150 and 400 mg/kg bw/day from days 6 through 19 of gestation.

The results showed that no animals died during the study. The number of females with live foetuses on gestation Day 20 was 24 in each of the control, low, mid- and high dose groups. No clinical signs of toxicological significance were noted during the study and no signs of reactions to treatment were observed during the dosing period. No differences of toxicological relevance were noted in body weight of females during the study, between control and treated groups. At body weight gain, a statistically significant increase of approximately 21% was noted on Day 6 post coitum in females receiving 400 mg/kg/day and a statistically significant decrease of approximatelly 29 and 67 % was noted on Day 9 post coitum in females receiving 150 and 400 mg/kg/day respectively, when compared to controls. These changes were considered of no toxicological relevance.

No relevant changes were detected in food consumption between treated and control females. No significant differences in terminal body weight, gravid uterus weight and absolute weight gain were observed in treated groups, when compared to the control group.

 Litter data, mean foetal weight and sex ratios were not affected by treatment. No treatment-related changes were observed at post mortem examination in treated animals, when compared to the controls.

Agnatia was detected in one foetus of the mid-dose group, one dead foetus was detected in the high dose group and small foetuses were detected in control and treated groups. All these findings were considered incidental and not dose-related.

Malformations were detected in control, low and mid-dose groups. Considering that some of these alterations were observed in small foetuses (foetal weight < 2.7 g) and were not dose-related, they were considered incidental.

No findings that could be considered treatment-related were noted at visceral examination of the foetuses in any group.

None of the litter parameters recorded (implantations, live fetuses, percentage of male/female fetuses, fetal body weight) were affected.

There were no treatment-related malformations and there were no treatment-related variations that were considered to be adverse. 

The maternal NOAEL was therefore 400 mg/kg bw/day.

The developmental NOAEL was therefore 400 mg/kg bw/day.

 

The effects of N-(3-DIMETHYLAMINOPROPYL) METHACRYLAMIDE on pregnancy and on embryo-foetal development were investigated in the rabbit, following oral administration (GLP study according to OECD 414). In-house mated females were randomly distributed to 4 groups with 25 females per group: 0/ 20/ 60/ 180 mg/kg bw/ day.

The test item was dissolved suspended in the vehicle (softened water) and administered from Day 6 through Day 28 post coitum at the dose volume of 10 mL/kg. Control animals received the vehicle alone during the same treatment period, at the same dose volume. Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean sectioned on Day 29 post coitum and subjected to post mortem examination. The number of corpora lutea, implantations, early and late intra-uterine deaths, live foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external and internal abnormalities. Fixed-soft head from approximately half of the foetuses (i.e. routinely, every second live foetus) and skeletal examinations were performed in all groups.

No mortality related to treatment occurred.

The number of females with live foetuses on gestation Day 29 was: 24 each in the control and high dose groups, 23 in the low dose group and 22 in the mid-dose group.

Isolated cases of hairloss, staining or damaged ear observed both in controls and/or treated females are not considered related to treatment.

No significant differences in body weight were seen. A statistically significant reduction in body weight gain was observed in high dose females receiving 180 mg/kg/day on Days 9 and 12 of gestation. Pregnant females of the mid-dose group receiving 60 mg/kg/day showed a reduction in absolute body weight gain in a single occasion, on gestation Day 9.

No effects were seen in low dose females receiving 20 mg/kg/day.

A statistically significant reduction in food consumption was observed in the high dose females compared to controls from Day 9 to 21 of gestation. A recovery was observed from Day 24 of gestation.

A reduction in uterusweight was noted in all treated females copared to controls but without a dose relation. This reduction was attributable to the lownumber of implantation observed the treated groups. The differences in number of implantation are not treatment related since animals were not treated during the implantation period (Days 0-5 of gestation).

The reduction in the number of live foetuses observed in the high dose group is linked to the decrease in implants that occurred before the start of treatment. Mean foetal weight was comparable between groups, the reduction in mean foetal weight observed in the high dose group is related to the low number of live foetuses.

No differences in sex ratios were seen between the control and the treated groups.

Macroscopic examination of dams: The findings observed in the two unscheduled dead rabbits were considered incidental and therefore not treatment-related. Animals killed at termination did not show relevant macroscopic changes. Agenesia of uterus (left horn) noted in one low dose female was consideres spontaneous in origin.

External and internal examinations of foetuses at necropsy: No treatment-related findings were described.

Foetal fixed head examination: No treatment related alteration in foetuses were described.

Skeletal examination did not show any treatment related findings.

Conclusion

Maternal toxicity

Signs of toxicity was present in the high dose animals receiving 180 mg/kg/day, demonstrated by a reduction of body weight gain on gestation Day 9 and 12. In addition, food consumption was reduced from Day 12 to Day 21 of gestation. These treatment related effects did not influence the pregnancy status of the animals, no animals aborted or were sacrificed during the study. Recovery from the observed effects started to be evident on Day 15 of gestation for body weight and from Day 24 of gestation for food consumption. As animals recovered in the later study phase, these effects were not considered as adverse. The uterine examinations did not reveal any effects on foetuses in term of early or late resorptions and foetal death. At necropsy all animals at termdid not show abnormalities that could be considered related to treatment. On the basis of this results the dosage of 180 mg/kg/day is considered the NOAEL for dams.

Developmental toxicity

The mean foetal weight of all treated groups was comparable to controls. No differences in sex ratioswere observed. At the external and internal examination of foetuses, no treatment related abnormalities were described. Skeletal and fixed head examination did not revealed any treatment related findings. On the basis of these results the dosage of 180 mg/kg/day is considered the NOAEL for foetuses.

Based on the results of these studies N-Dimethylaminopropyl methacrylamide shows no developmental toxic effects up to concentrations of 400 mg/kg/day in the rat and uo tp 180 mg/kg/d in the rabbit (detemined NOAELs).

Supporting information is provided by a reproductive/developmental toxicity screening study in rats (OECD 422). Based on parameters related to developmental toxicity in this study implantation loss, foetal growth and observation of gross malformations the NOAEL for developmental toxicity was 400 mg/kg bw/d, the highest dose tested.

Justification for selection of Effect on developmental toxicity: via oral route:
The studies have a valid reliability 1, GLP, guideline studies for developmental toxicity OECD 414, in which no relevant maternal toxicity up to the highest dose tested and no developmental toxicity was observed.

Justification for classification or non-classification

Based on the results of a Combined repeated dose toxicity study (OECD 422) with reproduction/developmental toxicity

N-[3-(dimethylamino)propyl)methacrylamide is not required to be classified for its toxicity to reproduction.