N,N-dimethylacrylamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

other: Reproduction / Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test (Jul 2000)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethylacrylamide

EC Number:

220-237-5

EC Name:

N,N-dimethylacrylamide

Cas Number:

2680-03-7

Molecular formula:

C5H9NO

IUPAC Name:

N,N-dimethylacrylamide

Details on test material:

- Name of test material (as cited in study report): N,N-Dimethylacrylamide
- BASF Test substance number: 11/0599-2
- Physical state/ appearance: liquid/colorless to yellowish, clear
- Batch: 20120615
- Purity: 99.4 corr. area-% (study No. 12L00359)
- Homogeneity: given (visually; study No. 12L00359)
- Storage: room temperature; under light exclusion, avoid temperatures >40°C
- Stability: the stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Expiry date: 30 Jun 2013

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age (when supplied): 11-12 weeks
- Weight at study initiation: Males: 330.6-337.3g, Females: 203.6-206.4g
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²). During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. Pregnant animals and their litters were housed together until PND 4 (end of lactation). Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Type NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, were added for environmental enrichment.
- Diet: Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, available ad libitum.
- Water: drinking water (from water bottles), available ad libitum.
- Acclimation period: 6 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- Amount of vehicle: 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Methods:
- Analysis of the test substance preparations: The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The study was carried out in compliance with the Principles of Good Laboratory Practice.
The stability of N,N-Dimethylacrylamide in drinking water at room temperature for a period of 7 days was demonstrated before the start of the administration period (BASF Project No. 01Y0599/11Y032). Homogeneity of N,N-Dimethylacrylamide was verified in the highest and lowest concentration. Additionally, concentration control was verified in all concentrations at the beginning of the study.
- Food analysis: The supplier assayed the food used in the study for chemical and microbiological contaminants.
- Drinking water analysis: the drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory.
- Bedding and enrichment analysis: The bedding and the enrichment are regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).
Results:
- Stability analyses: The stability of the test substance in drinking water was demonstrated over a period of 7 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.
- Homogeneity control analyses: Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that N,N-Dimethylacrylamide was distributed homogeneously in drinking water.
- Concentration control analyses: The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of 90-110% of the nominal concentrations. Taken together, the results demonstrate the correctness of the concentrations of N,N-Dimethylacrylamide.
- Food analyses: On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1×10^5/g food. Individual results can be found in the archives of the Experimental Toxicology and Ecology of BASF SE.
- Drinking water analyses: On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants. Individual results can be found in the archives of the Experimental Toxicology and Ecology of BASF SE.
- Bedding and enrichment analyses: On the basis of the analytical findings the bedding and the enrichment are found to be suitable. Levels given in Lab. Animal, Nov-Dec 1979, pp. 24-34, served as a guideline for maximum tolerable contaminants. Individual results are to be found in the archives of the Experimental Toxicology and Ecology of BASF SE.

Duration of treatment / exposure:

Males were exposed for 29 days, namely during 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females were exposed for 50 days, namely during 2 weeks prior to mating, during mating, gestation and 4 days of lactation, and then up to the day prior to scheduled necropsy.

Frequency of treatment:

Daily at the same time in the morning (exception: no administration to animals being in labor).

Doses / concentrations

Remarks:

Doses / Concentrations:
5, 15, 45 (first week)/30 (from day 7) mg/kg bw/day
Basis:
other: Actual ingested (Because of body weight loss in every single male and female animal in test group 3 during the first study week, the dose was reduced from 45 to 30 mg/kg bw/d from study day 7 onwards.)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Selection of doses: At the request of the sponsor.
- Method of formulation: N,N-Dimethylacrylamide was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were prepared daily. The administration volume was 10 mL/kg body weight.
- Mating procedures: In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group. The animals were paired by placing the female in the cage of the male mating partner from about 15.00 h until 07.00-09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".
- Parturition: The females were allowed to litter and rear their pups until day 4 after parturition. On PND 4, all pups were sacrificed and examined.

Examinations

Observations and examinations performed and frequency:

- Mortality / Viability: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
- Clinical signs: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal. The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of littering was considered the 24-hours period from about 15.00 h of one day until about 15.00 h of the following day.
- Food consumption: Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
Food consumption was not determined during the mating period (male and female F0 animals); Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14, 14-20; Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4. Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).
- Water consumption: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.
- Body weight data: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20; Females with litter were weighed on the day of parturition (PND 0) and on PND 4; Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- General reproduction and delivery data: The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs. Male and female mating fertility indices, gestation index and gestation length were calculated. The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters.

Sacrifice and pathology:

- Necropsy: All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.
- Terminal body weight and organ weights: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Epididymides, Testes.
- Gross pathology: The following organs or tissues were fixed in 4% buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Cervix, Coagulating glands, Epididymides (modified Davidson’s solution), Ovaries (modified Davidson’s solution), Oviducts, Prostate gland, Seminal vesicles, Testes (modified Davidson’s solution), Vagina, Uterus.
- Histopathology: Fixation was followed by histotechnical processing (HE staining), examination by light microscopy and assessment of findings at the follwing organs: Testes, Epididymides, Ovaries.

Other examinations:

Litter/pups:
- Pup number and status at delivery: All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.
- Pup viability/mortality: In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index PND 0-4 was calculated.
- Clinical observations: The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
- Body weights: The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
- Sex and sex ratio: On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth.
- Necropsy: All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-bycase basis, depending on the type of finding noted.

Statistics:

The following statistical methods were used to analyze the data:
- Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, implantation sites, pups delivered, life pups: Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means.
- Male and female mating indices, male and female fertility indices, gestation index, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups: Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (onesided) for the hypothesis of equal proportions.
- Mating days until day 0 pc: Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians.
-Viability Index: Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians.
- Terminal body weight and organ weights: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Results and discussion

Results of examinations

Details on results:

- Mortality: No parental animal died prematurely in the present study.
- Clinical signs: All animals of both sexes in test group 3 (45 and 30 mg/kg bw/d) showed poor general condition, closed eyelids, piloerection and reduced attention after the test substance administration at beginning of pre-mating phase. Furthermore, female animal Nos. 135 and 140 of test group 3 (45 and 30 mg/kg bw/d) showed piloerection after the test substance administration at the beginning of mating phase. In addition, female animal No. 135 showed piloerection shortly after the test substance application at the beginning of gestation phase. These findings were assessed as being related to treatment and adverse. No clinical signs of toxicity were observed for male and female animals of test groups 1 and 2 (5 and 15 mg/kg bw/d) over the entire study period.
- Food consumption: In male animals of test group 3 (45 and 30 mg/kg bw/d), food consumption was significantly decreased during the pre-mating period, i.e. between study days 0 and 7 as well as between study days 0 and 13. In female animals of test group 3 (45 and 30 mg/kg bw/d), food consumption was significantly decreased during the entire pre-mating period. The same animals showed reduced food consumption during the first week of gestation. These findings were assessed as being related to treatment and adverse. No impairment of food consumption was observed for male and female animals of test groups 1 and 2 (5 and 15 mg/kg bw/d) over the entire study period.
- Water consumption: No test substance-related findings were observed.
- Body weights: During the pre-mating phase, i.e. on study days 7 and 13, mean body weight was significantly decreased in both sexes of test group 3 (45 and 30 mg/kg bw/d); between study days 0 and 7 even a body weight loss occurred in both sexes. The same was true for the body weight change values, i.e. between study days 0 and 7 as well as between study days 0 and 13. Lower body weight change values were also observed in test group 2 (15 mg/kg bw/d), i.e. in male (significantly) and female (not significantly) animals during the premating period. During the entire gestation phase mean body weight of female animals of test group 3 (45 and 30 mg/kg bw/d) was significantly lower. On GD 14 the same was true for female animals of test group 2 (15 mg/kg bw/d).
At the beginning of lactation (PND 0) mean body weight of female animals of test group 3 (45 and 30 mg/kg bw/d) was significantly lower. The same was true for female animals of test group 2 (15 mg/kg bw/d). These findings were assessed as being related to treatment and adverse. No impairment of body weight parameters was observed for male and female animals of test group 1 (5 mg/kg bw/d) over the entire study period.
- Male reproduction data: For nearly all F0 parental males, which were placed with females to generate F1 pups, mating was confirmed, with the exception of male animal Nos. 23 of test group 2 (15 mg/kg bw/d; mated with female animal No 123) and 40 of test group 3 (45 and 30 mg/kg bw/d; mated with female animal No. 140). Thus, the male mating index was 100% in test groups 0 (0 mg/kg bw/d; control group) and 1 (5 mg/kg bw/d), 90% in test group 2 (15 mg/kg bw/d) and 3 (45 and 30 mg/kg bw/d). Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Each one male animal of test groups 0, 2 and 3 (0, 15 as well as 45 and 30 mg/kg bw/d; animal Nos. 5, 23 and 40 mated with female Nos. 105, 123 and 140, respectively) did not generate F1 pups. Thus, the male fertility index ranged between 90% and 100%. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Female reproduction and delivery data: The female mating index was 100% for test groups 0 and 1 (0 and 5 mg/kg bw/d) and 90% for test groups 2 and 3 (15 as well as 45 and 30 mg/kg bw/d). The mean duration until sperm was detected (GD 0) was 3.2, 3.6, 2.5 and 2.6 days in test groups 0-3 (0, 5, 15 as well as 45 and 30 mg/kg bw/d). The female fertility index was 90% for the control group and 100% for all groups given the test substance. The mean duration of gestation was between 22.0 and 22.4 days and did not show significant differences. The gestation index was 100% in test groups 0, 2 and 3 (0, 15 as well as 45 and 30 mg/kg bw/d) and 90% in test group 1 (5 mg/kg bw/d). Only one stillborn pup was seen in animal No. 125 of test group 2 (15 mg/kg bw/d; male pup No. 125-07), so the rate of liveborn pups was 99.2% in test group 2 (15 mg/kg bw/d) and 100% in test groups 0, 1 and 3 (0, 5 as well as 45 and 30 mg/kg bw/d). Postimplantation loss was not significantly changed when test groups 1-3 were compared to the control animals.
- Terminal body weight: When compared with control group 0 (set to 100%), terminal body weight was significantly decreased in one or more test groups (males group 3: 91%; females group 2: 94%; females group 3: 90%). These changes were regarded to be treatment-related.
- Organ weights: Mean absolute weights of the testes and epididymides did not show significant differences when compared to the control group 0. When compared with control group 0 (set to 100%), the relative weight of the testes was significantly increased (+11%) in males of test group 3 (45 and 30 mg/kg bw/d). The increase of the relative testis weight was related to the reduced terminal body weight in these animals.
- Gross lesions: All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment. Female animals Nos. 105, 123 and 140, which were not pregnant, as well as the male mating partners Nos. 5, 23 and 40, respectively, did not show gross lesions.
- Histopathology: All findings were considered to be incidental or spontaneous in origin and without any relation to treatment. Female animal Nos. 105 and 140, which were not pregnant as well as their male mating partners, i.e. Nos. 5, 40, did not show histopathological findings in the investigated organs. Female animal No. 123 and its male mating partner No. 23 were not investigated histopathologically.
- Litter data: Details on litter results are presented under 7.8.1 Toxicity to reproduction.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Treatment-related signs of systemic toxicity at 15 mg/kg bw/day (reduced body weight) and 45/30 mg/kg bw/day (reduced body weight and clinical signs).

Target system / organ toxicity

Critical effects observed:

not specified