N-(butoxymethyl)acrylamide

Carcinogenicity

Description of key information

The carcinogenicity of the read-across substance, acrylamide, was assessed via the oral route using methodology equivalent to OECD Test Guideline 453.  The NOAEL was ≥0.1 mg/kg bw/day for carcinogenic effects, however the tumours were not considered relevant to humans due to substantially different mammary gland physiology; the tunica vaginalis tumours appear specific for the F344 rat.

The carcinogenicity of the read-across substance, acrylamide, was assessed via the oral route using methodology equivalent to OECD Test Guideline 451. The NOAEL was 0.5 mg/kg bw/day (actual dose received) for both neurotoxic and carcinogenic effects.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 29, 1986 - March 11, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See the Analogue Approach Report attached in Section 13 of the IUCLID dossier.

Reason / purpose for cross-reference:

other: Read across to target substance

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

not specified

Principles of method if other than guideline:

The goals of this study were to:
- Better define the dose response characteristics of the central nervous system (CNS), mammary, and mouth tumours in female rats.
- Better characterize the dose response for induction of tunica vaginalis mesotheliomas and central nervous system tumours in male rats.
- Establish whether 0.1 mg/kg/day is a true NOEL for tunica vaginalis mesotheliomas.
- More accurately quantify background variability of tumours in males and females, especially CNS tumours in males.
- Determine the biological significance of the CNS tumours in the high dose males.
- Remove the potential bias of pair caging.
- Remove potential effects of SDA virus infection.
Because the design goals were different for the male compared to the female rats, it was decided to design each study independently of the other.

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Facility, Kingston, New York
- Age at study initiation: 44-45 days
- Weight at study initiation: male rats = 72-144 g , female animals = 75-122 g.
- Fasting period before study: No
- Housing: stainless steel suspended cage
- Diet: ad libitum : rodent chow, AGWAY PROLAB R-M-H 3200 Certified Rodent Meal (Chesapeake feed, Beltsville, MD)
- Water: ad libitum: Tap water was filtered through charcoal prior to mixing with the test substance
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): (72.6°F +2.1°F
- Humidity (%): 62.8 +7.3% relative humidity
- Air changes (per hr): 160 air changes per hour
- Photoperiod : 12 hrs dark /12 hrs light

IN-LIFE DATES: From: 1986-01-29; To: 1988-03-11 (terminal necropsy)

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was administered in the drinking water. Test solutions were prepared by adding an appropriate amount of test material into a small volume of charcoal filtered tap water to form a premix at a concentration of 20 mg/ml. An appropriate amount of this premix was then added to charcoal filtered tap water and the volume adjusted with additional charcoal filtered tap water to achieve the desired concentration. Fresh solutions were prepared weekly just prior to administration to the test animals. Excess solution needed to replace the contents of spilled bottles was stored at room temperature. Concentrations were determined based upon previous water consumption and body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the initiation of the study, a two-week stability study of acrylamide in dosed water was completed. In this stability study, acrylamide was added to deionised water to provide concentrations of 0.75, 3.75, 6.5, 15.0 or 19.5 ppm. Acrylamide concentration was analyzed by High Pressure Liquid Chromatography (HPLC). Samples were analyzed at the time of preparation and at 3, 7, 10, and 14 days after preparation and storage at room temperature or in the refrigerator. During the course of the study, acrylamide concentration in the drinking water was analyzed periodically. In addition, the acrylamide concentration was periodically measured in bottles removed from animal cages to document stability during the treatment period. Calculation of compound consumption during the first 16 weeks on study was done utilizing the target dose for each group, the water consumption for that individual animal and that animal's average body weight over that week. From week 17 until the end of the study, the prior body weight was used for the two weeks after a weighing and the next body weight was used for the week before a weighing.

Duration of treatment / exposure:

100-102 weeks

Frequency of treatment:

Continuous

Post exposure period:

None

Remarks:

Doses / Concentrations:
Males 0, 0.1, 0.5 and 2 mg/kg/day . Females 0, 1.0, and 3 mg/kg/day
Basis:
nominal in water
The concentration was adjusted to maintain a constant daily dose to the animals.

No. of animals per sex per dose:

Male Study:
(Control) 0 mg/kg/d = 102
(Control) 0 = 102
(Low Dose) 0.1 mg/mg/kg/d = 204
(Mid Dose) 0.5 mg/kg/d = 102
(High Dose) 2.0 mg/kg/d = 75

Female study:
(Control) 0 mg/kg/d = 50
(Control) 0 = 50
(Low Dose) 1mg/mg/kg/d = 100
(High Dose) 3.0 mg/kg/d = 100

An additional group of 25 males and 25 females were selected as sentinel animals.

Control animals:

yes, concurrent vehicle

Details on study design:

Male Dose Selection
Selection of doses to be tested in the males was driven by a desire to better define the background rate of CNS tumours in the control animals, clarify the dose response for the scrotal tumours, and to establish if o. 1 mg/kg/ day is a true no observed effect level for scrotal tumours. The only way to establish if 0.1 mg/kg/day was a statistically valid no observed effect level for scrotal tumours was to increase the number of animals tested so that a small increase in response could be statistically detected. An increase in the number of control animals was also be needed to better define the true background rate of CNS tumours. To achieve this goal, it was decided that the number of animals tested in the 0.1 mg/kg/day dose should be large enough to detect a 5% increase of tunica vaginalis mesotheliomas above the background tumour rate (1.3%) reported by Solleveld,(1984) for NTP studies. The same number of animals was also assigned to the control group. In order to evaluate the background variability of the CNS tumours it was also decided that the control group should be split into two groups. Designating two groups as controls allows statistical comparison to be made between the two groups for background variability of tumours. If little variability is seen, combining the two controls into a single control is justified. If extreme variability is seen, careful interpretation of statistically significant pairwise comparisons is needed. In addition to retesting the 0.1 mg/kg/day dose with more animals, it was also decided that the 0.5 and 2.0 mg/kg/day doses should be retested so as to clarify the scrotal and the unusual dose response mesothelioma dose response curve and the unusual dose response seen with the CNS tumours.
Female Dose Selection
An overriding feature of the acrylamide induced female tumours is that they only occurred in the highest dose tested (2.0 mg/kg/day). Because only one point was statistically elevated, predicting the shape of the dose response is tenuous and error prone. Ideally, therefore, doses higher and lower than 2.0 mg/kg/day should be tested. Testing a higher dose would also increase the probability of detecting a statistical increase for rarer tumours of the CNS, thyroid, uterus, mouth, and clitoris. However, 2.0 mg/kg/day is thought to represent a maximum tolerated dose (MTD) based on neurotoxicity. Clearly, retesting the previously tested doses of 0.01, 0.1 and 0.5 was unfounded because the expected incidence of the above tumours was far below the incidence which can be statistically detected with a reasonable number of animals. The above restraints led to a decision to bracket the 2.0 mg/kg/day dose by using 1.0 mg/kg/day and 3.0 mg/kg/day. It was felt that if individual caging of animals were used, the higher death rate seen after 21 months in the 2.0 mg/kg/day female rats would be avoided. Furthermore, close examination of the 2.0 mg/kg/day animals did not indicate any gross manifestations of neurotoxicity which might have caused life shortening. These considerations suggested 3.0 mg/kg/day was an acceptable MTD. In order to address the question of background variability for female tumours, two control groups were also used.
In summary, addition of a 3.0 mg/kg/day dose enhanced the characterization of the dose response and provided dose response confirmation for tumours that only occurred in the high dose. The 1.0 mg/kg/day dose provided an intermediate response point between the negative findings seen at the 0.5 mg/kg/day dose and the positive response seen at the 2.0 mg/kg/day dose in the Johnson, (1986) study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Twice daily. Cage side observations checked included mortality or moribundity and/or obvious signs of toxicity.
DETAILED CLINICAL OBSERVATIONS: For the first 16 weeks of the study, animals were removed from their cages and carefully examined for pharmacotoxic clinical signs and tissue masses once each week. From week 16 to week 40, these examinations were performed once every 4 weeks. From week 42 to the end of the study (week 106), these examinations were done once every two weeks.
BODY WEIGHT: The body weight of each animal on study was recorded prior to administration of the test material and then weekly through week 16. Body weights were recorded every 4 weeks thereafter until termination of the study at 106-108 weeks. Water consumption was measured weekly throughout the study. Feed consumption and water consumption data are not reported for those animals whose feed or water had spilled or whose water was inaccessible due to the tipping of the sipper tube.
FOOD CONSUMPTION Individual feed consumption was measured weekly during the first 16 weeks of the study and every 4 weeks thereafter until termination of the study.
FOOD EFFICIENCY - Not determined
WATER CONSUMPTION AND COMPOUND INTAKE: Water consumption was measured weekly throughout the study. Feed consumption and water consumption data are not reported for those animals whose feed or water had spilled or whose water was inaccessible due to the tipping of the sipper tube.

Sacrifice and pathology:

Complete postmortem gross pathologic examinations were performed on all rats in the study. Brain, liver, kidneys, and testes were excised and weighed. Group mean organ weights and organ-to-body weight ratios were calculated. Representative sections from all major organs and tissues (including the sciatic nerve) were stained with hematoxylin and eosin for histopathologic examination. Initially, microscopic examination was completed only on highdose and control rats. Based on histopathologic results in these groups, examinations were performed on specific tissues harvested from rats of lower dose groups. Histopathologic examination was performed on thyroid, brain (three levels, females only), mammary glands (females), and testes (males) in all rats. In addition, spinal cord (three levels), uterus, and gross lesions were evaluated in all control and high dose females, and in low dose female rats found dead or sacrificed moribund. Brain (three levels), spinal cord (three levels), and gross lesions were examined in all control and high-dose males and in low- and mid-dose male rats found dead or sacrificed moribund. No special staining methods were used to enhance light microscopic detection of degenerative changes in nervous tissues.

Other examinations:

At the terminal sacrifice, animals were fasted overnight and a terminal body weight was obtained. In addition, fresh organ weights of brain, liver, kidneys and testes were obtained. Paired organs (kidneys and testes) were weighed paired. Mean organ weights and organ-to-body weight ratios were determined.

Statistics:

Body weight, food consumption and water consumption were analysed by a one-way analysis of variance (ANOVA). If the analysis of variance indicated possible differences between control and compound-treated groups, Dunnett's t-test was used to determine which means of the compound-treated groups were significantly different from the controls at the 95% confidence interval. Statistical evaluations included comparisons of all groups relative to Control Group 1, all groups relative to Control Group 2, and all groups relative to the pooled control groups (Control groups 1 and 2 combined data). Pairwise t-tests were used to compare the mean absolute organ weights (and mean % relative organ weights) between the pooled control groups and each treated group by sex and organ.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Increased mortality at all doses in females and high dose in males

Mortality:

mortality observed, treatment-related

Description (incidence):

Increased mortality at all doses in females and high dose in males

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased bocy weight gain at the high dose males and both female groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Not applicable

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Decreased water consumption but copmpound intake was controlled.

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

No significant changes

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Peripheral neuropoathy at the high dose

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Presence of tumours complicated this observation

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Mammary tumours easily visible

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Nerve degeneration

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Tunica vaginalis and thyroid tumors in males and mammary gland and thyroid tumors in females.

Details on results:

At the level of behavioural and clinical observation performed in this bioassay protocol, no clinical signs of neurotoxicity were reported in any treated rats. Sciatic nerve degeneration was characterized by vacuolated nerve fibers of minimal-to-mild severity. Application of Fisher’s Exact test shows significantly increased incidences of sciatic nerve degeneration among both male and female high-dose rats. Histopathologic examination revealed significantly increased incidences of male thyroid gland (follicular cell) adenoma (and adenoma or carcinoma combined) and tunica vaginalis mesothelioma in the 2.0 mg/kg-day group. Females exposed to 1.0 and 3.0 mg/kg-day developed a significantly increased incidence of mammary gland fibroadenomas or combined fibroadenomas and carcinomas. Only the high-dose (3.0 mg/kgday) females exhibited a significantly increased incidence of thyroid gland follicular cell neoplasms (adenomas or carcinomas combined.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL concerns both neurotoxicity and carcinogenicity.

Conclusions:

Under the conditions of this study, there was a statistically significant increased incidence of scrotal mesotheliomas at the high dose of 2 mg/kg/day in males and of thyroid follicular cell adenomas of both males and females at the high dose of 2 and 3 mg/kg/day, respectively, and at the low dose in females (1 mg/kg/day). This study, combined with the data from a prior study, has shown that 0.5 mg/kg/day acrylamide in the drinking water is a no observed effect dose for both sexes for chronic administration of acrylamide to rats for both neurotoxicity and carcinogenicity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.5 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The key study was conducted on the read-across substance in vivo using methodology equivalent to an internationally recognised guideline and in compliance with GLP.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Statistically significant increases in thyroid gland (follicular cell) adenoma (and adenoma or carcinoma combined) and tunica vaginalis mesothelioma were observed in males. Statistically significantly increased incidence of mammary gland fibroadenomas or combined fibroadenomas and carcinomas were observed in females. Therefore the substance is classified as carcinogenic Category 1B.

Additional information

The carcinogenicity of the read-across substance, acrylamide, was assessed via the oral route using methodology equivalent to OECD Test Guideline 453 in male and female rats in a two-year study. The rats were exposed for their lifetimes to various concentrations of the read-across material in drinking water at 0.0, 0.01, 0.1, 0.5 and 2.0 mg/kg/day. Interim sacrifices were made at 6, 12 and 18 months. Extensive histological investigation on the central and peripheral nervous systems was performed. Observations included mortality, clinical signs, body weight, food and water consumption, clinical chemistry, haematology, urinalysis, gross pathology, organ weights, and histopathology. 

There were no significant adverse effects on gross pathology at 6, 12 and 18 months. However, at 24 months, there was an increase in the number of subcutaneous masses amongst females at 2 mg/kg/day. In males, there was a statistically significantly increased incidence of benign follicular cell adenomas of the thyroid at the highest dose level. In females there was a non-significant increase in the incidence of benign follicular cell adenomas of the thyroid and malignant adenocarcinomas.

In females there was a statistically significant increase in the incidence of malignant adenocarcinomas in the uterus.

In males there was a statistically significant increase in the incidence of tunica vaginalis mesothelioma at 0.5 and 2 mg/kg/day.

In males there was a non-significant increase in the incidence of malignant astrocytomas in the spinal cord.

There were also non-significant increases in malignant astrocytotomas in the brain of females, glial proliferation in the brain suggestive of an early tumor, and malignant astrocytomas in the spinal cord. In addition, malignant astrocytomas were also observed in the brain, and glial proliferation (suggestive of an early tumour). 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 and a non-significant increase in focal hyperplasia. The incidence of malignant carcinomas did not show any clear dose-response. For males, the incidence of tumour formation in the oral cavity did not show any clear exposure relationship although there was a statistically significant increase in focal hyperplasia of the hard palate.Again, although effects are not clear, there are some concerns as there is a possibility that hyperplasia and subsequent, but unclear, tumour 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 tumours of mammary glands, benign pituitary gland adenomas, and benign tumours of the clitoral gland. In males there were increased incidences of benign tumours in the adrenal glands (pheochromocytoma).

The increased incidences of mammary tumours, 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 tumours - 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. When evaluating a human relevance table, none of these tumours appear relevant to humans. Humans have substantially different mammary gland physiology from rodents and the tunica vaginalis tumours appear specific for the F344 rat. Only the thyroid may have significance, although there are no documented cases where chemicals have induced thyroid cancer in humans.

The NOAEL was ≥ 0.1 mg/kg bw/day based on induced tumours in the mammary gland, testes and thyroid.

In a second study, the carcinogenicity of the read-across substance was assessed via the oral route using methodology equivalent to OECD Test Guideline 451 in male and female rats. The test material was administered in the drinking water at 0, 0.1, 0.5 and 2 mg/kg/day for males and 0, 1.0 and 3 mg/kg/day for females.

Selection of doses to be tested in the males was driven by a desire to better define the background rate of CNS tumours in the control animals, clarify the dose response for the scrotal tumours, and to establish if 0.1 mg/kg/day is a true no observed effect level for scrotal tumours. The only way to establish if 0.1 mg/kg/day was a statistically valid no observed effect level for scrotal tumours was to increase the number of animals tested so that a small increase in response could be statistically detected. An increase in the number of control animals was also be needed to better define the true background rate of CNS tumours. To achieve this goal, it was decided that the number of animals tested in the 0.1 mg/kg/day dose should be large enough to detect a 5 % increase of tunica vaginalis mesotheliomas above the background tumour rate (1.3 %) reported by Solleveld (1984) for NTP studies. The same number of animals was also assigned to the control group. In order to evaluate the background variability of the CNS tumours it was also decided that the control group should be split into two groups. Designating two groups as controls allows statistical comparison to be made between the two groups for background variability of tumours. If little variability is seen, combining the two controls into a single control is justified. If extreme variability is seen, careful interpretation of statistically significant pairwise comparisons is needed. In addition to re-testing the 0.1 mg/kg/day dose with more animals, it was also decided that the 0.5 and 2.0 mg/kg/day doses should be retested so as to clarify the scrotal and the unusual dose response mesothelioma dose response curve and the unusual dose response seen with the CNS tumours.

An overriding feature of the female tumours induced by the read-across material is that they only occurred in the highest dose tested (2.0 mg/kg/day). Because only one point was statistically elevated, predicting the shape of the dose response is tenuous and error prone. Ideally, therefore, doses higher and lower than 2.0 mg/kg/day should be tested. Testing a higher dose would also increase the probability of detecting a statistical increase for rarer tumours of the CNS, thyroid, uterus, mouth, and clitoris. However, 2.0 mg/kg/day is thought to represent a maximum tolerated dose (MTD) based on neurotoxicity. Clearly, retesting the previously tested doses of 0.01, 0.1 and 0.5 was unfounded because the expected incidence of the above tumours was far below the incidence which can be statistically detected with a reasonable number of animals. The above restraints led to a decision to bracket the 2.0 mg/kg/day dose by using 1.0 mg/kg/day and 3.0 mg/kg/day. It was felt that if individual caging of animals were used, the higher death rate seen after 21 months in the 2.0 mg/kg/day female rats would be avoided. Furthermore, close examination of the 2.0 mg/kg/day animals did not indicate any gross manifestations of neurotoxicity which might have caused life shortening. These considerations suggested 3.0 mg/kg/day was an acceptable MTD. In order to address the question of background variability for female tumours, two control groups were also used.

Cage side observations were performed twice daily to check mortality or moribundity and obvious signs of toxicity. Individual feed consumption was measured weekly during the first 16 weeks of the study and every 4 weeks thereafter until termination of the study. Water consumption was measured weekly throughout the study. The body weight of each animal on study was recorded prior to administration of the test material and then weekly through week 16. Body weights were recorded every 4 weeks thereafter until termination of the study at 106-108 weeks.

Complete post-mortem gross pathologic examinations were performed on all rats in the study. Brain, liver, kidneys, and testes were excised and weighed. Group mean organ weights and organ-to-body weight ratios were calculated. Representative sections from all major organs and tissues (including the sciatic nerve) were stained with haematoxylin and eosin for histopathologic examination. Initially, microscopic examination was completed only on high dose and control rats. Based on histopathologic results in these groups, examinations were performed on specific tissues harvested from rats of lower dose groups. Histopathologic examination was performed on thyroid, brain (three levels, females only), mammary glands (females), and testes (males) in all rats. In addition, spinal cord (three levels), uterus, and gross lesions were evaluated in all control and high dose females, and in low dose female rats found dead or sacrificed moribund. Brain (three levels), spinal cord (three levels), and gross lesions were examined in all control and high-dose males and in low- and mid-dose male rats found dead or sacrificed moribund. No special staining methods were used to enhance light microscopic detection of degenerative changes in nervous tissues. At the terminal sacrifice, animals were fasted overnight and a terminal body weight was obtained. In addition, fresh organ weights of brain, liver, kidneys and testes were obtained. Paired organs (kidneys and testes) were weighed paired. Mean organ weights and organ-to-body weight ratios were determined.

No clinical signs of neurotoxicity were reported in any treated rats. Sciatic nerve degeneration was characterized by vacuolated nerve fibres of minimal-to-mild severity. Application of Fisher’s Exact test shows significantly increased incidences of sciatic nerve degeneration among both male and female high-dose rats. Histopathologic examination revealed significantly increased incidences of male thyroid gland (follicular cell) adenoma (and adenoma or carcinoma combined) and tunica vaginalis mesothelioma in the 2.0 mg/kg-day group. Females exposed to 1.0 and 3.0 mg/kg-day developed a significantly increased incidence of mammary gland fibroadenomas or combined fibroadenomas and carcinomas. Only the high-dose (3.0 mg/kg/day) females exhibited a significantly increased incidence of thyroid gland follicular cell neoplasms (adenomas or carcinomas combined).

The NOAEL was 0.5 mg/kg bw/day (actual dose received) for both neurotoxic and carcinogenic effects.