N,N'-methylenediacrylamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acrylamide

EC Number:

201-173-7

EC Name:

Acrylamide

Cas Number:

79-06-1

Molecular formula:

C3H5NO

IUPAC Name:

Prop-2-enamide

Details on test material:

- Name of test material (as cited in study report): Acrylamide
- Physical state: Colourless, odourless crystals
- Analytical purity: 96 to 99%
- Impurities (identity and concentrations): water
- Lot/batch No.: XS521, Lot K7, enzyme grade
- Stability under test conditions: Stable for 4 days.
- Storage condition of test material: The bulk sample was stored in a freezer. During use, the aliquots were stored in containers placed in desiccators in a refrigerator. Drinking water bottles were changed every 3-4 days to assure substance would not degrade.
- Other: Specific Gravity: 1.122 at 30°C
- Supplier: Eastman Organic Chemicals Company, Rochester, N.Y.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding laboratory, Portage, Michigan
- Age at study initiation: 4 weeks
- Weight at study initiation (g): Males 106-108 (±8); females 85-86 (±5)
- Fasting period before study: no
- Housing: stainless steel cages having wire mesh floors
- Diet: ad libitum (Purina's Certified Rodent Chow #5002, Ralston Purina Co., St. Louis, Missouri
- Water: ad libitum (water from the municipal water supply)
- Acclimation period: 11days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2
- Humidity (%): 40-60
- Air changes (per hr): approximately 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

Drinking water solutions were prepared twice per week (every 3 or 4 days) by serially diluting a water concentrate (premix) prepared from a measured amount of the recrystallized test aliquot dissolved in a known volume of tap water.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Since some loss of acrylamide was anticipated from the drinking water after use, analytical effort was concentrated on drinking water samples selected at random after having been on the cage for 4 days. An interfering peak (present from both control and treated rats in the water sample collected after 4 days on the cage) with the approximate retention time of acrylamide was noted for the first month of the study precluding accurate analyses during that period. Modifying the eluent to 10/90 methanol/water with 0.1M monobasic sodium phosphate eliminated the interference. This eluent was subsequently used on all samples taken after having been on the cage for 4 days. The frequency of analyses was adjusted to adequately establish the dose received by the test animals. Most of the analyses were conducted at the two lowest dose levels because other potential interferences would present the greatest potential to distort the results at these levels. Thus, clear definition of acrylamide level was considered most necessary at these levels. Additionally, Morden et al. (1981b) demonstrated slightly greater percentage of microbial degradation over a 7-day, period at the lower dose levels in their test system. For this study, the mean values ranged from 94% to 105% of targeted values for all dose levels over the course of the study. No acrylamide was detected in the 99 samples collected from control rats. Analyses were frequently conducted on various drinking water preparations over the course of the study. The samples were analyzed by HPLC using a Radial-Pak C18 column, water eluent and UV detector at 200 nm (Campbell and Hermnn, 1980). Analyses were obtained at the time a solution was prepared (day 0) from the dosing solutions used to fill the water bottles. For 190 samples taken over the course of the study from the dosing solutions (day 0) or the premix, the average was 99±7% of target concentration

Duration of treatment / exposure:

2 years

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0.0, 0.01, 0.1, 0.5, 2.0 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

90

Control animals:

yes, concurrent no treatment

Details on study design:

- General Study Design: Groups of 90 rats/sex/dose group were given either 0 (controls), 0.01, 0.1, 0.5, or 2.0 mg acrylamlde/kg body weight/day via the drinking water for up to 2 years.
- Dose selection rationale: The doses were selected based upon the 13-week study cited in Morden et al., 1981. It was anticipated that the highest dose level (2.0 mg/kg/day) would produce a peripheral neuropathy detectable either by light or electron microscopy but not to the extent of being manifested clinically.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

The rats were generally observed twice daily during the work week for overt signs of toxicity or changes in demeanour. These observations included the animals’ movement within the cage, the availability of food and water, wastage of feed and the response to the opening and closing of the cage. Routine monitoring on weekends and holidays was limited to the removal of dead animals and animal husbandry procedures required to ensure the availability of food and water.
Parameters monitored during the study included mortality, body weight, food consumption, water consumption, clinical observations, haematology, clinical chemistry, urinalysis, organ weights, gross and histopathology. All rats were examined approximately monthly after the first month for palpable masses. Individual body weights were recorded monthly from all rats

Sacrifice and pathology:

SACRIFICE: Ten rats/sex/dose group were randomly selected for interim sacrifices after 6, 12 or 18 months on study. This selection occurred about 3 weeks prior to the respective interim sacrifice; any rats dying spontaneously prior to the selection process were thus automatically excluded from the interim sacrifice and conversely included as part of the group of 60 rats/sex/dose group designated for the 2-year terminal sacrifice. The results of the interim sacrifices and related clinical determinations are reported separately (Gorzinski etl., 1984). The rats were started on test on 15 September 1980 and the terminal sacrifice was from 22-30 September 1982 (study days 738-746).
GROSS PATHOLOGY: Gross pathology, organ weights, and histopathology was performed on all animals Gross necropsy included the adrenal glands, auditory gland, aorta, bone, bone marrow, brain, cecum, cervix, coagulating glands, epididymides, esophagus, eyes, heart, liver, kidneys, large intestine, lacrimal glands, larynx, lymph node (mediastinal and mesenteric), lungs, mammary gland, ovaries, oviducts, pancreas, pituitary, prostate, salivary glands, seminal vesicles, skin, small intestine, spinal cord, spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus, vagina, and gross lesion.
HISTOPATHOLOGY:
Sections of tissues and organs (except the auditory sebaceous gland which was not routinely processed) were processed in a standard manner, embedded in paraffin, sectioned at 5-6µ, stained with hematoxylin and eosin, and examined histologically by a veterinary pathologist. 3 different peripheral nerves (brachial plexus, saphenous branch of the femoral nerve, and the tibial branch of the sciatic nerve) were examined from 10 randomly selected rats/sex/dose group. (the 10 being those from which blood samples were collected for clinical chemistry analyses). Tibial nerves, identified as the target organ, were examined on a "blind" basis from all rats over a short time interval. The nerves were subjectively graded into degrees of involvement based upon the number of focal degenerative lesions ("digestion chambers") present as follows: very slight - one to five digestion chambers; slight - six to fifteen digestion chambers, averaging less than one per high power (400X) field; moderate - numerous digestion chambers, often 2-3 or more per high power field; or severe - digestion chambers or overt loss of nerve fibres involving at least one-third of the fibers in the nerve. It should be emphasized that these grades refer to degree of histologic change and not clinical neuropathy. Five coronal sections of the brain were routinely processed. The spinal cord was examined at three different locations (cervical, thoracic, and lumbosacral) fro all rats as specified in the protocol. Additional sections, either from the same embedded block or from additional pieces of fixed tissue, were occasionally prepared and examined to further clarify a gross or histopathologic observation. Based on histopathologic examination, additional sections of testes and epididymides (generally 4 of each organ) were examined from all male rats. Special stains were only infrequently used to assist in diagnosing some lesions. A complete inventory of tissues examined histologically is included with the histopathology tables.

Statistics:

Statistical Analysis.
Body weights: collected either weekly or monthly were evaluated by analysis of variance for differences between groups. If the overall analysis of variance was significant Dunnett's t-test was used to identify statistically significant differences between experimental groups and their control.
Cumulative mortality: data were tabulated monthly and analyzed for overall differences by the Gehan-Wilcoxon test.
Histopathologic observations: treatment group comparisons of cumulative incidence were examined primarily by Fisher's Exact Probability Test. For observations with a control incidence of at least 6 percent, a Bonferonni correction for multiple treatment-control comparisons was applied. For observations below a 6 percent background incidence, the true false-positive statistical error rate is well below the nominal 0.05 level set for Fisher's test, so no multiple comparison corrections were used. In the absence of positive Fisher's test for a histopathologic lesion, the Cochran-Armitage test for linear trend in incidence was performed, if the requirements for a valid chi-square statistic were met.
Motality: Because there were significant differences in median survival times among treatment groups by the Gehan-Wilcoxon test, Fisher's test was supplemented by the mortal i ty-adjusted compari son methods of Peto (1980), where these tests were deemed informative and valid (by the chi-square criterion above).
Water and food consumption: not analyzed statistically.
Outlying values were identified by the sequential procedure described by Grubbs (1969). Outliers were identified but not excluded from the descriptive statistics calculated for the clinical laboratory data and the organ and body weight data from the terminal sacrifice. Outlying values were excluded from the values reported for body weight, food and water consumption. They were also excluded when mixing instructions were calculated.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Clinical observations disclosed little apparent difference between dose groups. On study day 210 some rats from all dose groups were noted to have excessive lacrimation and enlarged salivary glands consistent with sialodacryoadenitis virus (SOA) infection and the study room was quarantined by the clinical veterinarian. All groups, males and females, appeared to be equally affected. The swollen salivary glands resolved within a period of three days. Photophobia and excessive lacrimation persisted for about 10 days, with a declining incidence. There were no apparent treatment effects on mortality until the 21st month of the study. Up to that time, spontaneous deaths appeared to be random and the groups having the highest mortality were the males given 0.01 mg/kg/day and the females given 0.1 mg/kg/day. From 21 months until termination there was increased mortality in rats given 2.0 mg/kg/day such that by the end of the study there was significantly increased mortality for both sexes. Increased mortality was not present in any of the other treatment groups.

BODY WEIGHT AND WEIGHT GAIN
A slight decrease in bodyweight (up to 4%) was noted amongst males at 2 mg/kg/day

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no significant effects on food consumption at 6, 12 and 18 months.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
There were no significant effects on water consumption.

HAEMATOLOGY
There were no significant adverse effects on haematology.

CLINICAL CHEMISTRY
There were no significant adverse effects on blood biochemistry.

URINALYSIS
There were no significant adverse effects on urinalysis.

GROSS PATHOLOGY
There were no significant adverse effects on macroscopic pathology at 6, 12 and 18 months However, at 24 months, there was an increase in the number of subcutaneous and mammary gland masses amongst females at 2 mg/kg/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathologically, there were no abnormalities at 6 months. At examinations performed from 12 months onwards there was an increase in the incidence and severity of tibial nerve degeneration amongst males at 2 mg/kg/day and from 18 months onwards in females at 2 mg/kg/day (focal swelling of nerve fibres with fragmentation of myelin and axon, and the formation of vacuoles containing small round eosinophilic globules and macrophages). There were no clear changes amongst animals at lower exposure levels or amongst other peripheral nerve samples (saphenous branch of the femoral nerve and brachial plexus).

HISTOPATHOLOGY: NEOPLASTIC
In males, there was a statistically significantly increased incidence of benign follicular cell adenomas of the thyroid at the highest dose level (1/60, 0/58, 2/59, 1/59, 7/59). In females there was a non-significant increase in the incidence of benign follicular cell adenomas of the thyroid (0/58, 0/59, 1/59, 1/58, 3/60) and malignant adenocarcinomas (1/58, 0/59, 0/59, 0/58, 3/60). In females there was a statistically significant increase in the incidence of malignant adenocarcinomas in the uterus (1/60, 2/60, 1/60, 0/59, 5/60, or 1.7%, 3.3%, 1.7%, 0, 8.3%). The historical control range was stated to be 0-2.3%. In males there was a statistically significant increase in the incidence of malignant testicular mesothelioma at 0.5 and 2 mg/kg/day (3/60, 0/60, 7/60, 11/60, 10/60 or 5%, 0, 12%, 18%, 17%). The historical control incidence was 3.1% with a range of 2-6%.. In males there was a non-significant increase in the incidence of malignant astrocytomas in the spinal cord (1/60, 0/60, 0/60, 0/60, 3/60). There were also non-significant increases in malignant astrocytotomas in the brain of females (0/60, 1/60, 0/60, 0/60, 3/60), glial proliferation in the brain suggestive of an early tumour (0/60, 0/60, 0/60, 1/60, 3/60), and malignant astrocytomas in the spinal cord (1/60, 0/59, 0/60, 0/60, 3/61). In addition, malignant astrocytomas were also observed in the brain (3/60, 0/60, 0/60, 2/60, 2/60), and glial proliferation (suggestive of an early tumour) in 0/60, 0/60, 0/60, 1/60, 1/60. The effects in astrocytomas for brain and spinal cord in males and females do not show any clear dose-response but there are some concerns as these tumours are occurring in potential target tissues and, according to the authors, the concurrent control values may have been abnormally high so trends would not have been clear. Also, the group sizes used in this study may not have been sufficiently large enough to detect clear increases. Overall, because of these limitations, the toxicological significance of the presence of these astrocytomas in this study is unclear. For females, there was a statistically significant increase in the incidence of benign papillomas in the oral cavity at 2 mg/kg/day (0/60, 3/60, 2/60, 1/60, 7/61) and a non-significant increase in focal hyperplasia (1/60, 2/60, 1/60, 0/60, 4/61). The incidence of malignant carcinomas did not show any clear dose-response (0/60, 0/60, 0/60, 2/60, 1/61). For males, the incidence of tumour formation in the oral cavity did not show any clear exposure relationship (carcinomas 2/60, 0/60, 1/60, 0/60, 2/60, and papillomas 4/60, 7/60, 0/60, 5/60, 4/60) although there was a statistically significant increase in focal hyperplasia of the hard palate (0/60, 1/60, 1/60, 1/60, 4/60, 5/60). Again, although effects are not clear, there are some concerns as there is a possibility that hyperplasia and subsequent, but unclear, tumor formation may have arisen as a result of local effects due to the route of exposure employed. In females there were increases in benign and malignant tumors of mammary glands (10/60, 11/60, 9/60, 19/58, 23/61 and 2/60, 1/60, 1/60, 2/58, 6/61 respectively or 17%, 18%, 15%, 33%, 38% and 3%, 2%, 2%, 3%, 10%), benign pituitary gland adenomas (25/59, 30/60, 32/60, 27/60, 32/60 or 42%, 50%, 53%, 45%, 53%), and benign tumors of the clitoral gland (0/2, 1/3, 3/4, 2/4, 5/5). In males there were increased incidences of benign tumours in the adrenal glands (pheochromocytoma) (3/60, 7/59, 7/60, 5/60, 10/60 or 5%, 12%, 12%, 8%, 17%). The increased incidences of mammary tumors, benign pituitary adenomas and adrenal pheochromocytomas are of doubtful toxicological significance due to the poor dose-response and high historical control incidence (18% for benign mammary tumours, 2% for malignant mammary tumors - NTP data only, 28-47% for pituitary adenomas, 1-14% for pheoachromocytomas). For clitoral adenomas the total number of tissues examined was too small to draw any firm conclusions.

HISTORICAL CONTROL DATA (if applicable) high historical control incidence (18% for benign mammary tumours, 2% for malignant mammary tumors - NTP data only, 28-47% for pituitary adenomas, 1-14% for pheoachromocytomas).

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

0.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

neuropathology

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 mg/kg bw/day (nominal)

System:

peripheral nervous system

Organ:

other: peripheral nerves

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Ingestion of acrylamide induced neurotoxicity in F344 rats at doses ranging from 0.01-2.0 mg/kg/day. Testicular atrophy was observed in rats at elevated doses. The No Observed Adverse Effect Level (NOAEL) was determined to be 0.5 mg/kg in both sexes.