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Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
liquid
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

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: distilled water
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
Doses / concentrations
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).

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

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.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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.

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