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Carcinogenicity

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Description of key information

Acrylic acid showed no evidence of carcinogenicity in a 2-year drinking water study in Wistar rats up to the highest dose tested of 78 mg/kg bw/day. In two dermal carcinogenicity studies in three mice strains (C3H/HeJ, C3H/HeN Hsd BR and Hsd:(ICR)BR), the frequency of skin tumours was not elevated compared to the vehicle controls. In a 2-year (104-week) inhalation carcinogenicity study in B6D2F1/Crlj mice, and F344/DuCrlCrlj rats no increase in the incidence of tumors was observed in either sex,.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Acrylic acid
- Analytical purity: > 99 %
- Impurities (identity and concentrations): stabilized with 200 ppm MEHQ
- Test Substance No.: 82/380
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Wistar (Chbb = THOM (SPF))
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: 42 days old
- Weight at study initiation:
- male animals: 177 (156 - 198) g
- female animals: 141 (122 - 157) g
- Fasting period before study: no
- Housing: singly in Type DK III stainless steel wire cages
- Diet (ad libitum): ground Kliba 343 rat/mouse/hamster "A" food supplied by KLINGENTALMUHLE AG, CH-4303, Kaiseraugst, Switzerland
- Water (ad libitum): tap water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hours/ 12 hours
Route of administration:
oral: drinking water
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration control analyses of the aqueous solutions of the test substances were carried out at the beginning of the study and at 3, 6, 12, 18, 24 and 28 months. The determinations were carried out by gas chromatography.The actual concentrations in the test solutions were in the ranges 96 to 106, 94 to 103 and 92 to 102 % of the target concentrations of 120, 400 and 1200 ppm, respectively.
Duration of treatment / exposure:
26 (males) -28 (females) months
Frequency of treatment:
continuously
Post exposure period:
no
Dose / conc.:
120 ppm (analytical)
Remarks:
corresponding to approx. 8 mg/kg bw/d
Dose / conc.:
400 ppm (analytical)
Remarks:
corresponding to approx. 27 mg/kg bw/d
Dose / conc.:
1 200 ppm (analytical)
Remarks:
corresponding to approx. 78 mg/kg bw/d
No. of animals per sex per dose:
50
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
The doses were established on the basis of a subchronic toxicity study in which Fischer 344 rats received acrylic acid in doses of about 83, 250 or 750 mg/kg body weight for a period of 3 months in the drinking water (Bushy Run Research Center, 1980).
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day (Monday to Friday) or once a day (Saturdays, Sundays and public holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during the first three months, then every 4 weeks

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly during the first three months, at intervals of 3 months after the third month of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: The blood samplings and the subsequent analysis of the blood samples were carried out approx. 12, 18, 24 and 26 or 28 months after the beginning of administration.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: in the first 10 surviving animals per test group and sex; only control and highest dose were examined
- Parameters examined:
hemoglobin, erythrocytes, hematocrit, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration, thrombocytes, leukocytes

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were exsanguinated, dissected and the gross pathology was assessed. The exsanguinated animals, and the liver, kidneys, testes/ovaries, spleen, brain and adrenals were weighed and the relative organ weights were determined.

HISTOPATHOLOGY: Yes
The following organs/tissues were fixed in 4% formaldehyde solution:
- liver, kidneys, adrenals, heart, lungs, thymus, esophagus, jejunum, colon, urinary, bladder, salivary glands, aorta, eyes, brain, thyroids/parathyroids, stomach, ileum, rectum, uterus, representative lymph nodes, accessory genital organs, skin, femur with articular surface, buccal mucosa, cervical/
thoracic/ lumbar cord, nasal mucosa, spleen, testes/epididymides, ovaries, pituitary, trachea, duodenum, cecum, pancreas, sternum with marrow, peripheral nerve, skeletal muscles, female mammary gland, tongue, all gross lesions, bone marrow (femur), vagina, coagulation gland, mandibular lymph node.
Statistics:
Clinical examinations: The statistical significance of the clinical data (drinking water consumption and body weight) was determined using an analysis of variance (ANOVA) with subsequent DUNNETT's test.
Blood examinations: For statistical evaluation the t-test was used to compare the individual dose groups with the control group.
Clinical signs:
no effects observed
Description (incidence and severity):
The 3 doses (120, 400 and 1200 ppm) administered as addition to the drinking water did not lead in any of the animals participating in the study to clinical signs that could be associated with the test substance administered.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities attributable to an influence of the test substance during the period of time for which acrylic acid was administered.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The sporadically significant values which were observed in the males of the 400-ppm group (max. 6 %) were unrelated to the dose and within the biological range of variation. Although the initial weights were almost identical, the mean body weights of the female rats of the 120, 400 and 1200 ppm groups were higher than those of the control group throughout the study period. These increased values were even sporadically significant in the female rats of the 120- and 400-ppm groups; however, since these increases often varied at different time intervals and were not clearly dose-related, in many cases a substance-induced relevance cannot be attributed to this finding.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The drinking water consumption of the male and female rats of the 120, 400 and 1200 ppm groups showed no clearly substance-induced adverse effects in comparison to the control group. The amount of water consumed by the male and female animals of the 120 and 400 ppm groups was somewhat higher than that of the relevant control group for the most part of the study. By contrast, the drinking water consumption of the male and female rats of the 1200 ppm group was slightly reduced for the most part of the study from day 42 onward. The differences found were mainly slight and independent of the dose and are therefore assessed as being incidental in nature.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The erythrocyte, leukocyte and platelet counts as well as the determination of hemoglobin and the erythrocyte indices calculated from these parameters resulted in no variations to be associated with the test substance. Due to the plausibility criteria applied the individual significant variations are not regarded as being substance-related.
The 26 or 28-month administration period of 1200 ppm acrylic acid to rats via the drinking water did not result in any variations of the white blood cells both in the males and in the females that were attributable to the test substance administered. The morphological variations of the white blood cells are equally distributed among the control and the highest dose group. Thus, it is certain that the changes found in the 1200-ppm group are not substance-related. The morphological features that were noteworthy are probably incidental or due to age.
During the 26- or 28-month administration period, there were no variation in the red blood cells both of the male and female that were substance-related. The pathognomic changes to red blood cells found in individual animals of the control and highest dose groups are probably incidental or due to age.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
A number of necropsy findings were noted in rats that died or were killed in extremis and in rats sacrificed at the termination of the study. The type, incidence, and severity of these findings were similar in treated rats and controls.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
A number of non-neoptastic lesions were observed. Except for a slightly increased incidence of hepatoceIlular fatty change in males of the high dose, the type, incidence, and severity of these lesions were not considered to distinguish treated rats from controls.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
A total of 970 primary neoplasms were observed: 249 in group 0, 267 in group 1, 212 in group 2, 242 in group 3. From the 970 primary neoplasms seen in this study, 769 were benign (204, 214, 162, 189 in groups 0, 1, 2, and 3, respectively) and 201 wene malignant (45, 53, 50, 53 in groups 0,1, 2, and 3, respectively). The total number of rats with neoplasm was 388 (97, 98, 94, 99 in groups 0, 1, 2 and 3, respectively). The number of rats with more than one primary neoplasm was 287 (77, 72, 60, 73 in groups 0, 1, 2, and 3, respectively). The number of treated rats with metastases was considered to be similar to that of control rats. (group 0, 1, 2, 3: 0, 120, 400, and 1200 ppm)
Neoplastic lesions were primarily seen in endocrine and reproductive organs, skin, hemolymphoreticular system, mesenteric lymph nodes.
The most frequently observed neoplastic lesions were adenomas of the anterior pituitary gland, benign and malignant pheochromocytoma of the adrenal medulla, leydig cell tumours of the testes, fibroadenomas of the mammary glands, islet cell adenomas and carcinomas of the pancreas, theca granulosa cell tumours and luteomas of the ovaries, stromal polyps of the uterus, malignant lymphomas and fibrous histiocytomas of the hemolymphoreticular system, hemangiomas of the mesenteric lymph nodes, C-cell and follicular neoplasms of the thyroid glands, and various epithelial and mesenchymal neoplasms of the skin.
The incidence and organ distribution of the tumours found in the groups treated with acrylic acid for 26 or 28 months did not differ from those of the controls, and the authors consider the observed neoplastic changes spontaneous.
Key result
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
>= 78 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
sytemic toxicity
Effect level:
>= 78 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Critical effects observed:
no

Besides the marginal reduction in water consumption in both sexes of the highest dose group the administration of acrylic acid via the drinking water over a period of 26- or 28-months did not lead to any toxic changes attributable to the test substance administration. Furthermore it can be stated that, under the experimental conditions chosen, acrylic acid was not oncogenic in Wistar rats.

The extensive histopathological examination of the preserved tissues revealed that in all three treatment groups, the non-neoplastic tissue changes did not differ from those of the controls. In the males in the highest dose group (1200 ppm) there was a slightly increased incidence in hepatocellular fatty deposits in comparison with the other treatment groups and the controls, and a connection between this

effect and the administration of acrylic acid cannot be ruled out. All the other non-neoplastic tissue changes found were primarily of an inflammatory or degenerative nature, and were classified as spontaneous changes. The tumours observed were mainly located in the endocrinal and reproductive organs, the skin, the haemolymphoreticular system, and in the mesenterial lymph nodes. The most frequently observed neoplastic changes were anterior pituitary lobe adenomas, benign and malignant phaeochromocytomas of the medulla of the adrenal gland, testicular tumours originating from Leydig's cells, fibroadenomas of the mammary glands, islet cell adenomas and carcinomas of the pancreas, theca (granulosa) cell tumours and luteomas of the ovaries, polyps of the stroma of the uterus, malignant lymphomas and fibrous histiocytomas of the haemolymphoreticular system, haemangiosarcomas of the mesenterial lymph nodes, C-cell and follicular thyroid neoplasmas, and various neoplasmas of the dermal epithelia and mesenchyma. The incidence and organ distribution of the tumours found in the groups treated with acrylic acid for 26 or 28 months did not differ from those of the controls, and the authours consider that the neoplastic changes observed were spontaneous. Overall, no clear toxic or oncogenic effects were revealed by the histopathological examination of Wistar rats that had been administered acrylic acid at the maximum tolerable dose in the drinking water for 26 (males) or 28 (females) months.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
78 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
OECD TG 451

Carcinogenicity: via inhalation route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
only short summary report
Qualifier:
according to guideline
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Version / remarks:
adopted on May 12, 1981
GLP compliance:
yes
Species:
mouse
Strain:
other: B6D2F1/Crlj
Details on species / strain selection:
Male and female B6D2F1/Crlj mice (SPF) purchased from Charles River Laboratories Japan, Inc. (Atsugi Breeding Center, 795 Shimofurusawa, Atsugishi, Kanagawa) were used in the study.
227 each of male and female animals were introduced at the age of 4 weeks and subjected to quarantine and acclimation for 1 week each. From the animals, which are normal growth and showed no abnormalities in general conditions, 200 each of male and female animals with body weight close to the median were selected (a range of body weight at the time of group assignment, 21.7 to 25.4 g for males and 18.2 to 21.5 g for females) and used in the study.
The reason for selecting B6D2F1/Crlj mice (SPF) was that they are genetically stable, have data from many previous carcinogenicity studies, and the susceptibility to chemical-induced tumor development is known.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal assignment and animal identification
Animals without abnormalities in general conditions and changes in body weight were assigned to each group one by one in descending order of body weight. From the second round, animals were assigned in descending order of body weight to the group with lower body weight after a total body weight in each group was compared between groups so as to reduce bias in body weight among groups (proper stratification method).
Animals were identified by application of dye on the tail using an oil-based marker during the quarantine and acclimation periods and by ear punch during the treatment period. Each cage was labeled with an identification number.
Animals were housed in an independent room within the barrier area, and the door of the room was labeled with the study number, animal species, and animal numbers to distinguish from animals used in other studies and different animal species.

Rearing environment
Animals were kept in the quarantine room during the quarantine period and in an inhalation chamber in the inhalation laboratory during the acclimation and treatment periods.
The environmental conditions and cages used in the quarantine room, inhalation laboratory and inhalation chamber are shown below. Measured values (mean values ± standard deviation) of temperature and humidity in the quarantine room and inhalation laboratory are shown in < >. There were no significant changes in the environment in the quarantine room, inhalation laboratory or inhalation chamber that might affect the health of the animals.
Temperature: Quarantine room; 23 ± 2C
Inhalation laboratory; 22 ± 2C
Inhalation chamber; 23 ± 2C
Humidity: Quarantine room; 55 ± 15%
Inhalation chamber; 50 ± 20%
Light-dark cycle: 12 hours light (8:00 to 20:00)/12 hours dark (20:00 to 8:00)
Ventilation frequency: Quarantine room; 15 to 17 times/hour
Inhalation laboratory; 7 to 9 times/hour
Inhalation chamber; 12 ± 1 times/hour
Pressure: Inhalation chamber; 0 to -15 × 10 Pa
Housing of animals in cages: Single housing
Material, shape, size, etc. of a cage: Quarantine period; Stainless-steel, 2-chamber, mesh cages (112(W) × 212(D) × 120(H) mm/animal)
Acclimation period; Stainless-steel, 6-chamber, mesh cages (95(W) × 116(D) × 120(H) mm/animal)
Treatment period; Stainless-steel, 5-chamber, mesh cages (100(W) × 116(D) × 120(H) mm/animal)


Feed
CRF-1 solid diet (sterilized by 30 kGy-γ-ray irradiation, manufactured at Chiba Factory, Oriental Yeast Co., Ltd., 8-2 Shinminato, Mihama-ku, Chiba-shi, Chiba) was given ad libitum from a solid feed feeder throughout the rearing period. However, animals were not fed from the evening on the day before the scheduled necropsy.
As for the nutritional components of the feed used in the study, in-house analysis data for each lot used were obtained from Oriental Yeast Co., Ltd. and stored. As for contaminants in the feed, the analysis data of each lot used were obtained from the Japan Food Research Laboratories (52-1 Motoyoyogi-cho, Shibuya-ku, Tokyo) and Eurofins Scientific Co., Ltd. (4-16-21 Shimouma, Setagaya-ku, Tokyo), checked against the acceptance criteria specified in the protocol to confirm the absence of abnormalities and stored.

Drinking water
Animals were allowed free access to filtered, UV-irradiated municipal water (supplied by Hadano City Waterworks Bureau, Kanagawa) from an automatic watering system throughout the rearing period.
Drinking water was periodically sampled at the testing facility, tested at the Hadano Research Institute of Food and Drug Safety Center (729-5 Ochiai, Hadano, Kanagawa) and analyzed for specified items with reference to the Water Supply Law. The results were checked against the acceptance criteria specified in the study protocol to confirm that there was no abnormality, and stored.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
not specified
Remarks:
Animals were systemically exposed
Vehicle:
air
Details on exposure:
Animals were systemically exposed to the test substance by pouring air containing the test substance at a set concentration into the inhalation chamber where the test animals were kept.
The dosing concentrations were set at 3 levels of 2, 8 and 32 ppm (volume ratio, v/v) (a common ratio of 4). Clean air ventilation alone was used for the control group.

Gas generation method for the test substance and concentration adjustment
The test substance in the generator container of the supply device for the test substance (the special order item, Shibata Scientific Technology Ltd.) was heated in a circulation-type constant-temperature bath, and then evaporated by bubbling of clean air. After the vapor of this test substance was cooled to a constant temperature in a circulation-type constant-temperature bath, it was heated again while mixing with clean air (diluted air) to make a mixed gas of a constant concentration, and a fixed volume of it was supplied to the line mixer at the top of the inhalation chamber using a flow meter.
The concentration of the test substance in the inhalation chamber was monitored by a gas chromatograph, and the amount of the test substance supplied to the inhalation chamber was adjusted to the set concentration based on the concentration data.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Characteristics/Identity
The identity of the test substance was confirmed for each lot by measuring the mass spectrum with a mass spectrometer (Hitachi M-80 B) and the infrared absorption spectrum with an infrared spectrophotometer (Shimadzu FTIR-8200PC) and comparing them with their respective literature data.
As a result, the mass spectrum of the test substance showed the same molecular ion and fragment peaks as those in the literature data, and the infrared absorption spectrum showed peaks at the same wave numbers as those in the literature data. These results confirmed that the test substance was acrylic acid.

Stability
Each lot of the test substance was subjected to gas chromatography with a gas chromatograph (Agilent Technologies 5890 A) before and after use to confirm its stability by comparing the data.
As a result, there was no difference in measurement results for each lot before and after use, and it was confirmed that the test substance was stable during a period of use.

Measurement of test substance concentrations
The concentration of the test substance in the inhalation chamber was measured with a gas chromatograph (Shimadzu GC-14 B) equipped with an automatic sampling device every 15 minutes from the start to end of exposure.
For concentrations of the test substance in each group, the difference between the mean value and a set concentration ([mean value-set concentration]/set concentration × 100) was within 0.7%, and the coefficient of variation (standard deviation/mean value × 100) was within 1.3%. The results demonstrated that the concentrations in the chamber were precisely controlled.
Duration of treatment / exposure:
The duration of treatment was 6 hours a day, 5 days a week, for 104 weeks, in principle, for a total of 488 exposures.
Dose / conc.:
2 ppm (nominal)
Dose / conc.:
8 ppm (nominal)
Dose / conc.:
32 ppm (nominal)
No. of animals per sex per dose:
A total of 4 groups, 3 treatment groups and 1 control group, were established, and 50 animals per sex per group were used.
Control animals:
yes, concurrent no treatment
Details on study design:
Rationale for the route of administration, duration of treatment and dose
The route of administration was determined to be the transairway route by systemic exposure in accordance with the major exposure route to workers in the working environment where the test substance is produced and used.
The treatment period was 2 years (104 weeks) in accordance with the Standards for Investigation by Carcinogenicity Studies (Industrial Safety and Health Law) and OECD Guideline 451 for the Testing of Chemicals (Carcinogenicity Studies).
The doses were determined based on the results of the 2-week inhalation study and the 13-week study. The 2-week study was conducted at doses of 15 to 600 ppm (volume ratio v/v) (a common ratio of 2.5). As a result, all males and females died at 600 ppm group. No deaths occurred at 240 ppm or lower group, but the final body weight of males at 240 ppm group was less than 90% of final body weight of the control group. The 13-week study was conducted at doses of 0, 3.6, 10.7, 32, 96 and 180 ppm. As a result, no animal died probably due to the effect of acrylic acid, however, at 32 ppm or higher group, decreased body weight gain was mainly observed in males. Final body weights relative to the control group were 85% in males and 92% in females at 180 ppm group, 90% in males and 99% in females at 96 ppm group, and 90% in males and 96% in females at 32 ppm group. Histopathological changes in the nasal cavity were observed in both sexes in all treatment groups. However, based on the type and extent of the change in the nasal cavity, it was not considered to affect the survival of the animals. Since body weight gain was decreased by 10% in males at 32 ppm group as compared to the control group, the appropriate highest dose for the carcinogenicity study was considered to be 32 ppm. Since histopathological changes were observed in the nasal cavity of males and females up to the lowest dose of 3.6 ppm, it was considered appropriate to set the lowest dose in the carcinogenicity study at 2 ppm (TWA) which is the acceptable dose (ACGIH-TLV). Therefore, the highest dose for the carcinogenicity study was determined to be 32 ppm for both sexes, and the lower doses were set at 8 and 2 ppm (a common ratio of 4).
Observations and examinations performed and frequency:
Viability/mortality and clinical observations
Animals were observed for mortality and moribundity once daily and for detailed general conditions once a week.

Measurement of body weight
Body weight was measured once a week for 14 weeks after the start of treatment and every 4 weeks thereafter (also at Week 104). Animals were also weighed at the time when found dead, moribund sacrificed and transferred for the scheduled necropsy (body weight at the time of transfer).

Measurement of food consumption
The amount of food fed and the amount of remaining food were measured once a week for 14 weeks after the start of treatment and every 4 weeks thereafter (also at Week 104), the amount of food consumed per animal per day was calculated from these values.

Hematology
From surviving animals usable for blood collection at the time of the scheduled necropsy, blood was collected from the abdominal aorta under ether anesthesia immediately before necropsy into blood collection tubes containing EDTA-2 potassium and used for the following test items.
Test items: Red blood cell count, hemoglobin concentration, hematocrit value, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, reticulocyte ratio, white blood cell count and white blood cell differential

Blood biochemistry
From surviving animals usable for blood collection at the time of the scheduled necropsy, blood was collected from the abdominal aorta under ether anesthesia immediately before necropsy into blood collection tubes containing lithium heparin and centrifuged. The resulting plasma was used for the following test items.
Test items: Total protein, albumin, A/G ratio, total bilirubin, glucose, total cholesterol, triglyceride, phospholipid, AST, ALT, LDH, ALP, γ-GTP, CK, urea nitrogen, sodium, potassium, chloride, calcium, inorganic phosphorus

Urinalysis
Freshly voided urine was collected from surviving animals at the time of examination in Week 102 and was analyzed using urine test paper (Urolab Sticks, Siemens Healthcare Diagnostics, Ltd.) for the following test items.
Test items: pH, protein, glucose, ketone body, occult blood, urobilinogen
Sacrifice and pathology:
Necropsy
All animals were macroscopically observed.

Organ weight
Wet weights (actual organ weights) of the following organs from animals surviving until the scheduled necropsy were measured. The percentage of the wet weight of each organ to the body weight measured at the time of transfer (organ weight to body weight ratio) was calculated.
Organs measured: Adrenal gland, testis, ovary, heart, lung, kidney, spleen, liver, brain

Histopathology
The organs and tissues listed below were harvested from all animals, fixed in 10% neutral phosphate buffered formalin, embedded in paraffin, thin-sliced, stained with hematoxylin-eosin and histopathologically examined under a light microscope.
Organs and tissues examined: Skin, nasal cavity, nasopharynx, larynx, trachea, lung, bone marrow (femur), lymph node (axillary, abdominal wall, etc.), thymus, spleen, heart, tongue, salivary gland, esophagus, stomach, small intestine (including duodenum), large intestine, liver, gallbladder, pancreas, kidney, urinary bladder, pituitary gland, thyroid gland, parathyroid gland, adrenal gland, testis, epididymis, seminal vesicle, prostate gland, ovary, uterus, vagina, mammary gland, brain, spinal cord, peripheral nerve (sciatic nerve), eyeball, Harderian gland, muscle, bone (femur) and other organs and tissues with gross changes
The nasal cavity was resected (cross-sectioned) at 3 sites, i.e., the posterior edge of the incisor (Level 1), the incisor papilla (Level 2), and the anterior edge of the first molar (Level 3), and examined.
Statistics:
As for data of body weight, food consumption, hematology, blood biochemistry and organ weight, Bartlett's test was performed as a preliminary test to determine homogeneity of variance of the measured values using the control group as a reference group. If homogeneity of variance was found, a one-way ANOVA was performed. If a significant difference was found between groups, a test for mean values was performed using Dunnett's multiple comparison. If the variance was not equal, the measured values were ranked across the groups, and Kruskal-Wallis rank test was performed. If there was a significant difference between groups, Dunnett (type) multiple comparison was performed.
Non-neoplastic lesions in histopathological examination were classified into Grade 0 for animals without findings and Grade 1 to 4 for those with findings based on the severity and extent of lesions, and a χ2 test was performed. For urinalysis, a χ2 test was performed between the control group and each treatment group.
For neoplastic lesions, Peto test, Cochran-Armitage test and Fisher test were performed for the total number of tumor-bearing organs in each group for each organ tumor. The Peto test was performed using the contexts assigned at the time of histopathological examination, and the mortality method (test for tumors of the contexts 3 and 4), the prevalence method (test for tumors of the contexts 0, 1 and 2), and the mortality method + prevalence method (test for the total of the contexts 0 to 4) were performed.
Each test was conducted at a significance level of 5% (one-sided test for Peto test and Fisher's exact test, and two-sided test for other tests). The 5% and 1% significance levels were indicated when the test results were presented.
Clinical signs:
no effects observed
Description (incidence and severity):
- Males and Females -
There were no findings considered to be attributable to the effects of the test substance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Survival rates
- Male -
The survival rate was higher in the 32 ppm group than in the control group.
The number of surviving animals (survival rate) at Week 104 in each group was 26 (52%) at control group, 27 (54%) at 2 ppm group, 35 (70%) at 8 ppm group and 43 (86%) at 32 ppm group.
- Female -
There was no effect of the test substance on the survival rate of the treatment groups.
The number of surviving animals (survival rate) at Week 104 in each group was 19 (38%) at control group, 28 (56%) at 2 ppm group, 27 (54%) at 8 ppm group and 30 (60%) at 32 ppm group.

Cause of death
- Males and Females -
There was no increase in deaths due to specific lesions or tumors in the treatment groups.
In males at 32 ppm group, there were fewer deaths from urinary retention and liver tumors than in the control group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Male -
At 32 ppm group, decreased body weight gain was observed until the middle phase of the treatment period (Week 62), but body weight gain was recovered thereafter and changes in body weight were similar to those in the control group.
On the final day of measurement (Week 104), body weights relative to the control group were 95% at 2 ppm group, 97% at 8 ppm group and 102% at 32 ppm group.
- Female -
There were no changes considered to be attributable to the effects of the test substance.
On the final day of measurement (Week 104), body weights relative to the control group were 103% at 2 ppm group, 103% at 8 ppm group and 99% at 32 ppm group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Male -
At 32 ppm group, food consumption remained low almost throughout the treatment period. At 8 ppm group, no difference from the control group was observed in the early phase of treatment (up to Week 11), but low levels were observed in many weeks thereafter.
- Female -
At 32 ppm group, food consumption was low in some weeks during the treatment period. At 8 ppm group, food consumption was low in many weeks until the middle phase of treatment period (until Week 42), but after that, there was no difference from the control group.
Haematological findings:
no effects observed
Description (incidence and severity):
- Males and Females -
There were no changes considered to be attributable to the effects of the test substance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- Male -
At 32 ppm group, decreases in total cholesterol and ALT were observed.
- Female -
Decreased total bilirubin was observed at 8 ppm or higher group.
High triglyceride was observed at 8 ppm group, but it was not dose-dependent.
Urinalysis findings:
no effects observed
Description (incidence and severity):
- Males and Females -
There were no changes considered to be attributable to the effects of the test substance.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- Male -
At 8 ppm or higher group, decreased actual weight of the liver was observed.
- Female -
There were no changes considered to be attributable to the effects of the test substance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- Males and Females -
There was no increase in findings that might be attributable to the effect of the test substance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Male -

Increases in the lesions were observed in the olfactory epithelium, respiratory epithelium and glands.
In the olfactory epithelium, increases in the number of incidence and severity of respiratory epithelial metaplasia were observed by dose-dependent in all treatment groups. Respiratory epithelial metaplasia of the olfactory epithelium represented a change caused by replacement of the olfactory epithelium with ciliated respiratory epithelium. The severity of lesions was enhanced and more extensive changes in the olfactory epithelium were observed in the treatment groups than in the control group. In animals with mild lesions, respiratory epithelial metaplasia was observed only in the dorsal olfactory epithelium at either Level 2 or 3. In animals with moderate or severe lesions, changes in the dorsal olfactory epithelium were observed at both Level 2 and Level 3, and in addition to the dorsal side, changes in the ethmoturbinate were observed at Level 3. Increases in the number of incidence and severity of eosinophilic change in the olfactory epithelium were observed in all treatment groups. In addition, an increase in atrophy of the olfactory epithelium was observed at 8 ppm or higher group. The atrophy of the olfactory epithelium represented a change caused by a decrease in the number of olfactory cells resulting in a decrease in the height of the olfactory epithelium, which was mostly observed in the dorsal olfactory epithelium at Level 2.
Increases in the number of incidence and severity of eosinophilic changes in the respiratory epithelium were observed by dose-dependent in all treatment groups.
In the glands in the lamina propria, increases in the number of incidence and severity of respiratory epithelial metaplasia were observed in all treatment groups, and moderate to severe lesions were increased in the treatment groups depending on the dose. Respiratory epithelial metaplasia of the glands represented a change caused by replacement of the submucosal glandular tissue with the ciliated respiratory epithelium, and was mainly observed in the submucosa of the olfactory epithelium where atrophy and respiratory epithelial metaplasia were observed.
At 32 ppm group, ulcers and squamous metaplasia of the respiratory epithelium and exudates were observed in a few animals.


Increases in the number of incidence of eosinophilic change in the epithelium were observed at 32 ppm group.

The number of incidence of extramedullary hematopoiesis in spleen was significantly reduced at 32 ppm group.

- Female -

In the nasal cavity, increases in lesions were observed in the olfactory epithelium, respiratory epithelium and glands in the lamina propria.
Increases in the number of incidence and severity of respiratory epithelial metaplasia and eosinophilic changes in the olfactory epithelium were observed by dose dependent in all treatment groups.
Increases in the number of incidence of hyperplasia and eosinophilic change in the respiratory epithelium were observed at 32 ppm group. Respiratory epithelial hyperplasia was noted at the base of the nasal septum at Level 1, with no cellular or structural atypia.
In the glands in the lamina propria, increases in the number of incidence and severity of respiratory epithelial metaplasia were observed in all treatment groups, depending on the dose.
Increases in the number of incidence of exudates were observed at 32 ppm group.
At 32 ppm group, hyperplasia of the glands, squamous metaplasia of the respiratory epithelium and atrophy of the olfactory epithelium were observed in a few animals.


Increases in the number of incidence of eosinophilic change in the epithelium were observed in all treatment groups.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- Male -
No increase in the incidence of tumors attributable to treatment with the test substance was observed.

The Fisher test showed an increase in the incidence of Harderian gland adenoma at 32 ppm group (0% at control group, , 4% at 2 ppm group, 8% at 8 ppm group, 10% at 32 ppm group), however the incidence in this group was within the range of the historical control data (the lowest, 0%, to the highest, 10%; the mean incidence rate, 4.9%) and did not show an increase in the trend test. Therefore, the increased incidence of Harderian gland adenoma was considered not to be caused by exposure to the test substance.
In addition, the Fisher test showed a decrease in the incidence of hepatocellular carcinoma at 8 ppm and 32 ppm group, and the Cochran-Armitage test showed a decreasing trend.. However, the incidences in these groups were within the range of the historical control data (the lowest, 2%, to the highest, 42%; mean, 18.5%), and they were not considered to be caused by treatment with the test substance. Furthermore, the Fisher test showed a decrease in the combined incidence of hemangioma and hemangiosarcoma in the liver at 2 ppm group, but these changes were not dose-dependent.

- Female -
No increase in the incidence of tumors attributable to exposure to the test substance was observed.
Dose descriptor:
NOAEC
Remarks:
Systemic effects
Effect level:
2 ppm (nominal)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEC
Remarks:
Carcinogenicity
Effect level:
32 ppm (nominal)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
The 2-year (104-week) inhalation carcinogenicity study of acrylic acid was conducted in B6D2F1/Crlj mice, and the following conclusions were obtained.
No increase in the incidence of tumors was observed in either sex, and acrylic acid was not carcinogenic to mice.
Executive summary:

A 2-year inhalation study of acrylic acid in mice by systemic exposure (doses: 0 [control], 2, 8 and 32 ppm) was conducted. As a result, no neoplastic lesions were induced by acrylic acid.

 

Survival rate, clinical observations, body weight and food consumption

The survival rate was higher in males at 32 ppm group than in the control group. In males at 32 ppm group, the incidence of hepatocellular carcinoma was decreased, and deaths from liver tumors were decreased (control, n = 9; 32 ppm, n = 0). Furthermore, the number of deaths due to urinary retention was decreased (control, n=6; 32 ppm, n=0). Therefore, the survival rate was high. There was no effect of exposure to acrylic acid on general conditions.

In males at 32 ppm group, decreased body weight gain was observed until the middle phase of the treatment period (Week 62), but body weight gain was recovered thereafter, and changes in body weight were similar to those in the control group. Final body weights of males relative to the control group were 95% at 2 ppm group, 97% at 8 ppm group and 102% at 32 ppm group. In females of the treatment groups, changes in body weight were similar to those in the control group. Final body weights of females relative to the control group were 103% at 2 ppm group, 103% at 8 ppm group and 99% at 32 ppm group.

Food consumption remained low in males at 32 ppm group almost throughout the treatment period . At 8 ppm group, no difference from the control group was observed in the early phase of treatment (up to Week 11), but low levels were observed in many weeks thereafter. In females at 32 ppm group, food consumption was low in some weeks during the treatment period. At 8 ppm group, food consumption was low in many weeks until the middle phase of treatment period (up to Week 42), but after that, there was no difference from the control group.

 

Neoplastic lesions and tumor-associated lesions

No increase in the incidence of tumors and increase in the incidence of tumor-related lesions due to exposure to acrylic acid was observed in males and females.

The doses in the present carcinogenicity study were determined based on the results of the 2-week inhalation study and the 13-week inhalation study. In the 2-week study, all males and females died at 600 ppm group, but no deaths occurred at 240 ppm or lower group. The 13-week study was conducted at doses of 0, 3.6, 10.7, 32, 96 and 180 ppm. As a result, at 32 ppm or higher group, decreased body weight gain was observed mainly in males. Final body weights relative to the control group were 85% in males and 92% in females at 180 ppm group, 90% in males and 99% in females at 96 ppm group, and 90% in males and 96% in females at 32 ppm group. Histopathological changes in the nasal cavity were observed in males and females in all treatment groups. However, based on the type and extent of the change in the nasal cavity, it was not considered to affect the survival of the animals. Since body weight gain was decreased by 10% in males at 32 ppm group as compared to the control group, the appropriate highest dose for the carcinogenicity study was considered to be 32 ppm.

In the present carcinogenicity study, there was no decrease in survival rate due to exposure in both males and females at the highest dose of 32 ppm group. Histopathological changes were observed in the nasal cavity, but the final body weights relative to the control group were 102% in males and 99% in females. Therefore, the highest dose of 32 ppm in the present study could be below the above criteria for MTD. However, in males at 32 ppm group, body weight gain was decreased until the middle phase of the treatment period. Body weight was significantly lower than that in the control group at Weeks 4, 5, 7, and 9 to 62 of the treatment period. Considering together with the histopathological changes in the nasal cavity, which might be toxic effects of acrylic acid, the dose of 32 ppm in males is considered to be close to the MTD.

 

Other Effects

No changes were observed in hematology, urinalysis or necropsy. In blood biochemistry and organ weight measurement, changes were observed only in some parameters at 8 ppm or higher group.

Blood biochemistry revealed decreases in total cholesterol and ALT in males at 32 ppm group and decreased total bilirubin in females at 8 ppm or higher group. Organ weight measurement revealed decreased actual weight of the liver in males at 8 ppm or higher group. However, histopathology revealed no changes that might be related to changes observed in blood biochemistry and organ weight measurement.

Histopathology revealed changes in the nasal cavity and nasopharynx in both sexes, and increases in the incidence of respiratory epithelial metaplasia of the olfactory epithelium and glands and eosinophilic changes in the olfactory epithelium and respiratory epithelium. The incidence of atrophy of the olfactory epithelium in males and atrophy of exudate and hyperplasia of the respiratory epithelium in females were also increased. The number of incidence of eosinophilic changes in the nasopharynx was increased in both males and females.

In the olfactory epithelium, atrophy was observed in males as a change indicating damage, and respiratory epithelial metaplasia was observed in males and females as a change accompanied by the damage. Respiratory epithelial metaplasia have been reported to be observed as the repairing tissue of olfactory epithelium that was damaged. In addition, eosinophilic changes in the olfactory epithelium, respiratory epithelium and epithelium of nasopharynx of the nasal cavity observed in males and females have been reported to occur more frequently in old rats as age-related changes. Eosinophilic changes in the nasal cavity have been reported to occur after inhalation exposure to irritating chemical substances such as tobacco, chlorine and dimethylamine. The increased severity of eosinophilic changes observed in the nasal cavity and nasopharynx of animals exposed to acrylic acid in the present study was considered attributable to enhancement of eosinophilic changes as an age-related change by exposure to acrylic acid and irritation by acrylic acid. Hyperplasia of the respiratory epithelium was observed in females. This change was considered not to result in tumors because it was a proliferative lesion but did not show cellular atypia and structural atypia.

In the 13-week study conducted at our center as a preliminary study for the present study, changes in the nasal cavity were observed up to the lowest dose of 3.6 ppm. Changes in the nasal cavity were necrosis, atrophy, atrophy of the olfactory nerve fiber bundle, respiratory epithelial metaplasia and eosinophilic change in the olfactory epithelium, eosinophilic change in the respiratory epithelium, and respiratory epithelial metaplasia of glands in the lamina propria. In addition, eosinophilic changes in the nasopharynx were observed. Similarly to the 13-week study, the results of the present study showed the effects of exposure to acrylic acid on the olfactory epithelium, respiratory epithelium and glands in the lamina propria of the nasal cavity and nasopharynx. Changes in the nasal cavity were observed up to 2 ppm in both sexes.

There were no treatment-related effects on organs other than the nasal cavity.

 

Lowest Observed Adverse Effect Level (LOAEL)

As described above, in the present carcinogenicity study, blood biochemistry, organ weight measurement and histopathological examination revealed changes considered from the effect of acrylic acid.

Of these findings, respiratory epithelial metaplasia of the olfactory epithelium and glands in lamina propria of the nasal cavity and eosinophilic changes in the olfactory epithelium and respiratory epithelium in males, and respiratory epithelial metaplasia of the olfactory epithelium and glands in lamina propria of the nasal cavity, eosinophilic changes in the olfactory epithelium and eosinophilic changes in the nasopharynx in females were observed up to the lowest dose of 2 ppm in the histopathological examination. Therefore, the lowest observed adverse effect level (LOAEL) for 2-year inhalation exposure of mice to acrylic acid was considered to be 2 ppm based on the effects on the nasal cavity and nasopharynx as endpoints.

Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
only short summary report
Qualifier:
according to guideline
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
GLP compliance:
yes
Species:
rat
Strain:
Fischer 344/DuCrj
Details on species / strain selection:
Male and female F344/DuCrlCrlj rats (SPF) purchased from Charles River Laboratories Japan, Inc. (Atsugi Breeding Center, 795 Shimofurusawa, Atsugi-shi, Kanagawa) were used in the study.
227 each of male and female animals were introduced at the age of 4 weeks and subjected to quarantine and acclimation for 1 week each. From the animals, which are normal growth and showed no abnormalities in general conditions, 200 each of male and female animals with body weight close to the median were selected (a range of body weight at the time of group assignment, 114 to 134 g for males and 91 to 104 g for females) and used in the study.
The reason for selecting F344/DuCrlCrlj rats (SPF) was that they are genetically stable, have data from many previous carcinogenicity studies, and the susceptibility to chemical-induced tumor development is known.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal assignment and animal identification
Animals without abnormalities in general conditions or changes in body weight were assigned to each group one by one in descending order of body weight. From the second round, animals were assigned in descending order of body weight to the group with lower body weight after a total body weight in each group was compared between groups so as to reduce bias in body weight among groups (proper stratification method).
Animals were identified by application of dye on the tail using an oil-based marker during the quarantine and acclimation periods and by ear punch during the treatment period. Each cage was labeled with an identification number.
Animals were housed in an independent room within the barrier area, and the door of the room was labeled with the study number, animal species, and animal numbers to distinguish from animals used in other studies and different animal species.

Rearing environment
Animals were kept in the quarantine room during the quarantine period and in an inhalation chamber in the inhalation laboratory during the acclimation and treatment periods.
The environmental conditions and cages used in the quarantine room, inhalation laboratory and inhalation chamber are shown below. Measured values (mean value ± standard deviation) of temperature and humidity in the quarantine room and inhalation laboratory are shown in < >. There were no significant changes in the environment in the quarantine room, inhalation laboratory or inhalation chamber that might affect the health of the animals.
Temperature: Quarantine room; 23 ± 2C
Inhalation laboratory; 22 ± 2C
Inhalation chamber; 23 ± 2C
Humidity: Quarantine room; 55 ± 15%
Inhalation chamber; 50 ± 20%
Light-dark cycle: 12 hours light (8:00 to 20:00)/12 hours dark (20:00 to 8:00)
Ventilation frequency: Quarantine room; 15 to 17 times/hour
Inhalation laboratory; 7 to 9 times/hour
Inhalation chamber; 12 ± 1 times/hour
Pressure: Inhalation chamber; 0 to -15 × 10 Pa
Housing of animals in cages: Quarantine period; housed in groups (n = 5), Acclimation/treatment period; housed individually
Material, shape, size, etc. of a cage: Quarantine period; Stainless-steel, group-housing, mesh cages (340(W) × 294(D) × 176(H) mm/5 animals)
Acclimation period; Stainless-steel, 6-chamber, mesh cages (125(W) × 216(D) × 176(H) mm/animal)
Treatment period; Stainless-steel, 5-chamber, mesh cages (150(W) × 216(D) × 176(H) mm/animal)

Feed
CRF-1 solid diet (sterilized by 30 kGy-γ-ray irradiation, manufactured at Chiba Factory, Oriental Yeast Co., Ltd., 8-2 Shinminato, Mihama-ku, Chiba-shi, Chiba) was given ad libitum from a solid feed feeder throughout the rearing period. However, animals were not fed from the evening on the day before the scheduled necropsy.
As for the nutritional components of the feed used in the study, in-house analysis data for each lot used were obtained from Oriental Yeast Co., Ltd. and stored. As for contaminants in the feed, the analysis data of each lot used were obtained from the Japan Food Research Laboratories (52-1 Motoyoyogi-cho, Shibuya-ku, Tokyo) and Eurofins Scientific Co., Ltd. (4-16-21 Shimouma, Setagaya-ku, Tokyo), checked against the acceptance criteria specified in the protocol to confirm the absence of abnormalities and stored.

Drinking water
Animals were allowed free access to filtered, UV-irradiated municipal water (supplied by Hadano City Waterworks Bureau, Kanagawa) from an automatic watering system throughout the rearing period.
Drinking water was periodically sampled at the testing facility, tested at the Hadano Research Institute of Food and Drug Safety Center (729-5 Ochiai, Hadano, Kanagawa) and analyzed for specified items with reference to the Water Supply Law. The results were checked against the acceptance criteria specified in the study protocol to confirm that there was no abnormality, and stored.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
not specified
Remarks:
Animals were systemically exposed
Vehicle:
air
Details on exposure:
Animals were systemically exposed to the test substance by pouring air containing the test substance at a set concentration into the inhalation chamber where the test animals were kept.
The dosing concentrations were set at 3 levels of 10, 40 and 160 ppm (volume ratio, v/v) (a common ratio of 4). Clean air ventilation alone was used for the control group.

Gas generation method for the test substance and concentration adjustment
The test substance in the generator container of the supply device for the test substance (the special order item, Shibata Scientific Technology Ltd.) was heated in a circulation-type constant-temperature bath, and then evaporated by bubbling of clean air. The vapor of the test substance was mixed with clean air (conveyed air), further cooled to a certain temperature in a circulation-type constant-temperature bath, and re-heated and prepared at a certain concentration. Then, a certain volume was supplied to the line mixer at the top of the inhalation chamber using a flow meter.
The concentration of the test substance in the inhalation chamber was monitored by a gas chromatograph, and the amount of the test substance supplied to the inhalation chamber was adjusted to the set concentration based on the concentration data.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Characteristics/Identity
The identity of the test substance was confirmed for each lot by measuring the mass spectrum with a mass spectrometer (Hitachi M-80 B) and the infrared absorption spectrum with an infrared spectrophotometer (Shimadzu FTIR-8200PC) and comparing them with their respective literature data.
As a result, the mass spectrum of the test substance showed the same molecular ion and fragment peaks as those in the literature data, and the infrared absorption spectrum showed peaks at the same wave numbers as those in the literature data. These results confirmed that the test substance was acrylic acid.

Stability
Each lot of the test substance was subjected to gas chromatography with a gas chromatograph (Agilent Technologies 5890 A) before and after use to confirm its stability by comparing the data.
As a result, there was no difference in measurement results for each lot before and after use, and it was confirmed that the test substance was stable during a period of use.

Measurement of test substance concentrations
The concentration of the test substance in the inhalation chamber was measured with a gas chromatograph (Shimadzu GC-14 B) equipped with an automatic sampling device every 15 minutes from the start to end of exposure.
For concentrations of the test substance in each group, the difference between the mean value and a set concentration ([mean-set concentration]/set concentration × 100) was within 2.0%, and the coefficient of variation (standard deviation/mean × 100) was within 1.0%. The results demonstrated that the concentrations in the chamber were precisely controlled.

Duration of treatment / exposure:
The duration of treatment was 6 hours a day, 5 days a week, for 104 weeks, in principle, for a total of 487 exposures.
Dose / conc.:
10 ppm (nominal)
Dose / conc.:
40 ppm (nominal)
Dose / conc.:
160 ppm (nominal)
No. of animals per sex per dose:
A total of 4 groups, 3 treatment groups and 1 control group, were established, and 50 animals per sex per group were used.
Control animals:
yes, concurrent no treatment
Details on study design:
Rationale for the route of administration, duration of treatment and dose
The route of administration was determined to be the transairway route by systemic exposure in accordance with the major exposure route for workers in the working environment where the test substance is produced and used.
The treatment period was 2 years (104 weeks) in accordance with the Standards for Investigation by Carcinogenicity Studies (Industrial Safety and Health Law) and OECD Guideline 451 for the Testing of Chemicals (Carcinogenicity Studies).
The doses were determined based on the results of a 2-week inhalation study and a 13-week study. The 2-week study was conducted at doses of 38 to 600 ppm (volume ratio v/v) (a common ratio of 2). As a result, one female died at 600 ppm group. No deaths occurred at 300 ppm or lower group, but the final body weight of males at 300 ppm group was less than 90% of that of the control group. The 13-week study was conducted at doses of 0, 10, 24, 60, 150 and 200 ppm. As a result, no deaths occurred in any group. Final body weights of males relative to the control group were 90% at 200 ppm group and 94% at 150 ppm group. Histopathological changes in the nasal cavity were observed in both sexes in both groups. However, the type and severity of changes in the nasal cavity were not significantly different between these groups. Based on these results, 160 ppm, which is close to 150 ppm between 200 ppm and 150 ppm, was considered to be appropriate as the highest dose in the carcinogenicity study. Histopathological changes were observed in the olfactory epithelium of the nasal cavity in males and females up to 24 ppm group in the low-dose groups, but no changes were observed in the nasal cavity at 10 ppm. Thus, the appropriate lowest dose in the carcinogenicity study was considered to be 10 ppm. Therefore, the highest dose for the carcinogenicity study was determined to be 160 ppm for both sexes, and the lower doses were set at 40 and 10 ppm (a common ratio of 4).
Observations and examinations performed and frequency:
Viability/mortality and clinical observations
Animals were observed for mortality and moribundity once daily and for detailed general conditions once a week.

Measurement of body weight
Body weight was measured once a week for 14 weeks after the start of treatment and every 4 weeks thereafter (also at Week 104). Animals were also weighed at the time when found dead, moribund sacrificed and transferred for the scheduled necropsy (body weight at the time of transfer).

Measurement of food consumption
The amount of food fed and the amount of remaining food were measured once a week for 14 weeks after the start of treatment and every 4 weeks thereafter (also at Week 104), and the amount of food consumed per animal per day was calculated from these values.

Hematology
From surviving animals usable for blood collection at the time of the scheduled necropsy, blood was collected from the abdominal aorta under ether anesthesia immediately before necropsy into blood collection tubes containing EDTA-2 potassium and used for the following test items.
Test items: Red blood cell count, hemoglobin concentration, hematocrit value, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, reticulocyte ratio, white blood cell count and white blood cell differential

Blood biochemistry
From surviving animals usable for blood collection at the time of the scheduled necropsy, blood was collected from the abdominal aorta under ether anesthesia immediately before necropsy into blood collection tubes containing lithium heparin and centrifuged. The resulting plasma was used for the following tests.
Test items: Total protein, albumin, A/G ratio, total bilirubin, glucose, total cholesterol, triglyceride, phospholipid, AST, ALT, LDH, ALP, γ-GTP, CK, urea nitrogen, creatinine, sodium, potassium, chloride, calcium, inorganic phosphorus

Urinalysis
Freshly voided urine was collected from surviving animals at the time of examination in Week 100 and was analyzed using urine test paper (Multistix, Siemens Healthcare Diagnostics, Ltd.) for the following test items.
Test items: pH, protein, glucose, ketone body, bilirubin, occult blood, urobilinogen

Sacrifice and pathology:
Necropsy
All animals were macroscopically observed.

Organ weight
Wet weights (actual organ weights) of the following organs from animals surviving until the scheduled necropsy were measured. The percentage of the wet weight of each organ to the body weight measured at the time of transfer (organ weight to body weight ratio) was calculated.
Organs measured: Adrenal gland, testis, ovary, heart, lung, kidney, spleen, liver, brain

Histopathology
The organs and tissues listed below were harvested from all animals, fixed in 10% neutral phosphate buffered formalin, embedded in paraffin, thin-sliced, stained with hematoxylin-eosin and histopathologically examined under a light microscope.
Organs and tissues examined: Skin, nasal cavity, nasopharynx, larynx, trachea, lung, bone marrow (femur), lymph node (axillary, abdominal wall, etc.), thymus, spleen, heart, tongue, salivary gland, esophagus, stomach, small intestine (including duodenum), large intestine, liver, pancreas, kidney, urinary bladder, pituitary gland, thyroid gland, parathyroid gland, adrenal gland, testis, epididymis, seminal vesicle, prostate gland, ovary, uterus, vagina, mammary gland, brain, spinal cord, peripheral nerve (sciatic nerve), eyeball, Harderian gland, muscle, bone (femur) and other organs and tissues with gross changes
The nasal cavity was resected (cross-sectioned) at 3 sites, i.e., the posterior edge of the incisor (Level 1), the incisor papilla (Level 2), and the anterior edge of the first molar (Level 3), and examined.
Statistics:
As for data of body weight, food consumption, hematology, blood biochemistry and organ weight, Bartlett's test was performed as a preliminary test to determine homogeneity of variance of the measured values using the control group as a reference group. If homogeneity of variance was found, a one-way ANOVA was performed. If a significant difference was found between groups, a test for mean values was performed using Dunnett's multiple comparison. If the variance was not equal, the measured values were ranked across the groups, and Kruskal-Wallis rank test was performed. If there was a significant difference between groups, Dunnett (type) multiple comparison was performed.
Non-neoplastic lesions in histopathological examination were classified into Grade 0 for animals without findings and Grade 1 to 4 for those with findings based on the severity and extent of lesions, and a χ2 test was performed. For urinalysis, a χ2 test was performed between the control group and each treatment group.
For neoplastic lesions, Peto test, Cochran-Armitage test and Fisher test were performed for the total number of tumor-bearing organs in each group for each organ tumor. The Peto test was performed using the contexts assigned at the time of histopathological examination, and the mortality method (test for tumors of the contexts 3 and 4), the prevalence method (test for tumors of the contexts 0, 1 and 2), and the mortality method + prevalence method (test for the total of the contexts 0 to 4) were performed.
Each test was conducted at a significance level of 5% (one-sided test for Peto test and Fisher's exact test, and two-sided test for other tests). The 5% and 1% significance levels were indicated when the test results were presented.
Clinical signs:
no effects observed
Description (incidence and severity):
- Males and Females -
There were no findings considered to be attributable to the effects of the test substance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Survival rates
- Male -
There was no effect of the test substance on the survival rate of the treatment groups.
The number of surviving animals (survival rate) at Week 104 in each group was as follows: 38 (76%) at 0 ppm (control), 36 (72%) at 10 ppm, 41 (82%) at 40 ppm, and 42 (84%) at 160 ppm.
- Female -
There was no effect of the test substance on the survival rate of the treatment groups.
The number of surviving animals (survival rate) at Week 104 in each group was as follows: 37 (74%) at 0 ppm (control), 40 (80%) at 10 ppm, 38 (76%) at 40 ppm and 43 (86%) at 160 ppm.


Cause of death
- Males and Females -
There was no increase in deaths due to specific lesions or tumors in the treatment groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Male -
At 160 ppm, decreased body weight gain was observed throughout the treatment period.
On the final measurement day (Week 104), body weights relative to the control group were 98% at 10 ppm group, 98% at 40 ppm group and 87% at 160 ppm group.
- Female -
At 160 ppm group, decreased body weight gain was observed throughout the treatment period.
On the final measurement day (Week 104), body weights relative to the control group were 104% at 10 ppm group, 100% at 40 ppm group and 90% at 160 ppm group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Male -
At 160 ppm group, food consumption remained low throughout the treatment period. At 40 ppm group, food consumption was low mainly during the first half of the treatment period. At 10 ppm group, food consumption was low during the early period of treatment.
- Female -
At 160 ppm group, the food consumption was continuously low from Weeks 1 to 22 of treatment, and remained low thereafter although not significant in some weeks. At 40 ppm group, food consumption was mostly low during the first half of treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- Male -
At 160 ppm group, low MCV and MCH were observed.
- Female -
At 160 ppm group, high eosinophil percentage was observed in white blood cell differential.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- Male -
At 160 ppm group, increased A/G ratio and decreased total cholesterol, triglyceride, phospholipid and creatinine were observed.
At 10 ppm group, decreased total protein was observed, but the change was not dose-dependent.
- Female -
At 160 ppm group, increased A/G ratio and urea nitrogen were observed.
At 10 ppm group, increased triglyceride and phospholipid were observed, but these changes were not dose-dependent.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- Male -
At 160 ppm group, decreased positive protein levels were observed.
- Female -
At 40 ppm or higher group, increases in positive ketone bodies were observed.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- Male -
There were no changes considered to be attributable to the effects of the test substance.
At 160 ppm group, decreases in the actual weight of the heart, lung, kidney, spleen and liver and increases in the organ-body weight ratio of the heart, lung and brain were noted. These changes were considered due to decreased body weight at the time of transfer at 160 ppm group.
- Female -
There were no changes considered to be attributable to the effects of the test substance.
At 10 ppm group, decreased organ-body weight ratio of the lung was observed. However, this change was considered to be of no toxicological significance because there was no correlation with the dose or significant change in the actual weight of the lung. At 160 ppm group, increases in the organ-body weight ratio of the adrenal gland, heart, lung, kidney, liver and brain were observed. These changes were considered due to the low body weight at the time of transfer at 160 ppm group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- Males and Females -
There was no increase in findings that might be attributable to the effect of the test substance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Male -

Increased lesions were observed in the respiratory epithelium, olfactory epithelium and gland.
In the respiratory epithelium, increased number of incidence of squamous metaplasia was observed at 160 ppm group, and the severity of lesions was mild in most animals. Squamous metaplasia of the respiratory epithelium represented a change where the respiratory epithelium was replaced with squamous epithelium and was observed at Level 1, the tip of the turbinate near the front of the nose, the upper part of the maxillary turbinate and the nasal septum. Increases in the number of incidence of epithelial atrophy and disarrangement in the olfactory epithelium were observed at 40 ppm or higher group. Increases in the number of incidence of respiratory epithelial metaplasia and squamous metaplasia were observed at 160 ppm group. All of these lesions were mild in severity. Increased severity of eosinophilic changes in the olfactory epithelium was observed at 160 ppm group. The number of animals with moderate or severe eosinophilic changes was 30 animals at 160 ppm group, compared with 6 animals in the control group. The atrophy of the olfactory epithelium represented a change caused by a decrease in the number of olfactory cells resulting in a decrease in the height of the olfactory epithelium, which was observed in the dorsal side at Level 2 or Level 3. The disarrangement of the olfactory epithelium represented a change caused by disorganized arrangement of olfactory cells, which was observed in the dorsal side at Level 3 or the olfactory epithelium in the ethmoturbinate. Respiratory epithelial metaplasia of the olfactory epithelium represented a change caused by replacement with the respiratory epithelium of the olfactory epithelium, which was observed in the dorsal side at Level 2 or Level 3. Squamous metaplasia of the olfactory epithelium represents a change caused by replacement of the olfactory epithelium with squamous epithelium. The squamous metaplasia observed in the present study was replaced with epithelium consisting of prickle cells with an intercellular bridge, although formation of a keratinized layer was not observed. Squamous metaplasia of the olfactory epithelium was observed in the dorsal side at Level 2. Eosinophilic changes represent accumulation of eosinophilic droplets in the cytoplasm of the respiratory epithelium and olfactory epithelium. There was no difference in the affected site between the control and treatment groups. Increases in the number of incidence of respiratory epithelial metaplasia in the glands in the lamina propria were observed at 160 ppm group, and all lesions were mild in severity. The respiratory epithelial metaplasia of the glands represented a change in which the glands in the lamina propria were replaced with the respiratory epithelium and was observed in the dorsal side at Level 1 and in the septum, nasal gland or olfactory gland in the dorsal side at Level 2.

In addition, the severity of chronic nephropathy was reduced at 160 ppm group.
The incidence of ulcers in the forestomach at 10 ppm group was statistically significantly different from that in the control group, but the difference was not dose-dependent.

- Female -

Increased lesions were observed in the respiratory epithelium, olfactory epithelium and gland.
Increases in the number of incidence of squamous metaplasia in the respiratory epithelium were observed at 160 ppm group, and all lesions were mild in severity. Increases in the number of incidence of epithelial atrophy and disarrangement in the olfactory epithelium were observed at 40 ppm or higher group. Increases in the number of incidence of respiratory epithelial metaplasia and squamous metaplasia were observed at 160 ppm group. All of these lesions were mild in severity. Increased severity of eosinophilic changes was observed at 160 ppm group. The number of animals with severe eosinophilic change was 26 animals at 160 ppm group, compared with 8 animals in the control group. Increases in the number of incidence of respiratory epithelial metaplasia in the glands in the lamina propria were observed at 160 ppm group, and all lesions were mild in severity.

In addition, the inflammation of the nasolacrimal duct was decreased at 160 ppm group.


Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- Male -
No increase in the incidence of tumors attributable to exposure to the test substance was observed.
The combined incidence of hepatocellular adenoma and hepatocellular carcinoma in the liver was statistically significantly decreased at 40 ppm or lower group.
Although a statistically significant increase in the incidence of interstitial cell tumor of testicle was observed at 40 ppm group, the increase was not dose-dependent.

- Female -
No increase in the incidence of tumors attributable to exposure to the test substance was observed.
Key result
Dose descriptor:
NOAEC
Remarks:
Carcinogenicity
Effect level:
160 ppm (nominal)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEC
Remarks:
Systemic toxicity
Effect level:
10 ppm (nominal)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
Conclusions:
The 2-year (104-week) inhalation carcinogenicity study of acrylic acid was conducted in F344/DuCrlCrlj rats, and the following conclusions were obtained.
No increase in the incidence of tumors was observed in either sex, and acrylic acid was not carcinogenic to rats.
Executive summary:

A 2-year inhalation study of acrylic acid in rats by systemic exposure (doses: 0 [control], 10, 40 and 160 ppm) was conducted. As a result, no neoplastic lesions were induced by acrylic acid.

 

Survival rate, clinical observations, body weight and food consumption

No effects of exposure to acrylic acid on survival rate and general conditions were described. As non-neoplastic lesions, atrophy, respiratory metaplasia, squamous cell metaplasia, disarrangement of the olfactory epithelium, and squamous metaplasia of the respiratory epithelium, respiratory metaplasia of the gland in the nasal cavity were significantly increased in both sexes at doses of 40 ppm acrylic acid and above.

At 160 ppm group, decreased body weight gain was observed in males and females. Final body weights of males relative to the control group were 98% at 10 ppm group, 98% at 40 ppm group and 87% at 160 ppm group. Final body weights of females relative to the control group were 104% at 10 ppm group, 100% at 40 ppm group and 90% at 160 ppm group.

 

Based on the local effects in the nasal cavity and the reduced body weight gain there are indications that at 160 ppm the MTD might have been exceeded. OECD Guidance Document No. 116 on the Design and Conduct of Chronic Toxicity and Carcinogenicity Studies supporting OECD 451, 452 and 453 and OECD Guidelines for the Testing of Chemicals: Carcinogenicity Studies (OECD 451, 2018) state „The testing of substances at potentially irritating or corrosive concentrations/doses should be avoided, as administering such substances could result in severe pain and tissue damage at point-of-entry, which would compromise both animal welfare and the integrity of the study.“(OECD 116) which is supported by the OECD 451 recommending „Unless limited by the physical-chemical nature or biological effects of the test substance, the highest dose level should be chosen to identify the principal target organs and toxic effects while avoiding suffering, severe toxicity, morbidity, or death. While taking into account the factors outlined in paragraph 23 below, the highest dose level should normally be chosen to elicit evidence of toxicity, as evidenced by, for example, depression of body weight gain (approximately 10%).“(OECD 451)

Food consumption was continuously low throughout the treatment period in males and females at 160 ppm group. The food consumption was lower in males and females at 40 ppm group during the first half of the treatment period, and also lower in males at 10 ppm group in some weeks during the early treatment period. Decreased body weight gain in males and females at 160 ppm was considered to be associated with decreased food consumption.

Neoplastic lesions and tumor-associated lesions

No increase in the incidence of tumors and increase in the incidence of tumor-related lesions due to exposure to acrylic acid was observed in males and females.

The doses in the present carcinogenicity study were determined based on the results of the 2-week inhalation study and the 13-week inhalation study. In the 2-week study, one female died at 600 ppm group, but no deaths occurred at 300 ppm or lower group. The 13-week study was conducted at doses of 10 to 200 ppm. As a result, no animal died in any group. At 200 ppm group, histopathology revealed changes in the nasal cavity in males and females, and decreased body weight gain in males (90% of body weight gain in the control group). At 150 ppm group, histopathology of the nasal cavity revealed changes, however there was no difference in body weight in males or females. Based on these results, the highest dose in the carcinogenicity study was set at 160 ppm for both sexes.

In the present carcinogenicity study, final body weights relative to the control group were 87% in males and 90% in females, at 160 ppm group, the highest dose, and histopathological changes in the nasal cavity were observed. No decrease in survival rate due to exposure was observed in males and females. Therefore, the set of doses as the highest dose of 160 ppm for the present carcinogenicity study met/exceeded the above criteria for MTD.

Other Effects

Hematology and blood biochemistry revealed changes in some parameters at 160 ppm group. Urinalysis revealed changes in males at 160 ppm group and females at 40 ppm or higher group.

Hematology revealed decreased MCV and MCH in males and increased eosinophil percentage by white blood cell differential in females at 160 ppm group. Blood biochemistry revealed increased A/G ratio in males and females and decreases in total cholesterol, triglyceride, phospholipid and creatinine in males at 160 ppm group. Urinalysis revealed decreased positive protein levels in males at 160 ppm group and increased positive ketone bodies in females at 40 ppm or higher group. However, there were no changes in other hematology parameters including red blood cell count and pathology parameters that were considered to be related to these changes.

Necropsy and organ weights showed no changes attributable to acrylic acid.

Histopathology revealed the effects of acrylic acid on the nasal cavity of males and females. The number of incidences of atrophy, respiratory epithelial metaplasia, squamous metaplasia and disarrangement in the olfactory epithelium, squamous epithelial metaplasia of the respiratory epithelium and respiratory epithelial metaplasia of the glands of lamina propria was increased. The number of incidences of atrophy and disarrangement of the olfactory epithelium was increased in both males and females at 40 ppm or higher group. The number of incidences of other lesions in the nasal cavity was increased at 160 ppm group. As described above, atrophy was observed in the olfactory epithelium as a change indicating damage, and respiratory epithelial metaplasia, disarrangement and squamous metaplasia were observed as changes accompanied by the damage. It is reported that respiratory epithelial metaplasia and disarrangement observed as the repairing tissue of olfactory epithelium that was damaged. Squamous metaplasia of the olfactory epithelium represents a change accompanied by the repair of damaged tissue and chronic inflammation. Squamous metaplasia with atypia may appear as a preneoplastic lesion that progresses to tumors after inhalation exposure to chemical substances. In squamous metaplasia observed in the present study, changes indicating of proliferative lesions such as pathologically obvious cellular atypia and increased mitotic figures were not observed, and tumors derived from squamous epithelium did not occur. Increased incidence of eosinophilic changes in the olfactory epithelium has been reported as an age-related change in old rats. The increased severity of eosinophilic changes in the olfactory epithelium at 160 ppm group compared to the control group is considered attributable to age-related changes enhanced by exposure to acrylic acid. Eosinophilic changes in the olfactory epithelium have been reported to occur after inhalation exposure to irritating chemical substances such as tobacco chlorine and dimethylamine, however, their toxicological significance is presently unknown. In the respiratory epithelium, squamous metaplasia was observed as a change accompanied by damage. Squamous metaplasia has been reported to be observed accompanied by the repair of damage and inflammation. Although the results of the present study showed no changes indicating damage to the respiratory epithelium, necrosis of the respiratory epithelium was observed at 150 ppm group in the 2-week study of acrylic acid. Therefore, it is considered that in the present study, only reparative changes were observed as chronic effects after the respiratory epithelium was damaged in the early phase of treatment. No sign indicating proliferative lesions such as obvious cellular atypia and increased mitotic figures was observed in squamous metaplasia of the respiratory epithelium as a chronic effect, and no occurrence of tumors derived from squamous epithelium was observed. Respiratory epithelial metaplasia of glands (nasal gland, olfactory gland) in the lamina propria is reported to occur responsive to stimuli and increase with age. The number of incidences of the respiratory epithelial metaplasia of the glands was increased at 160 ppm group, enhancement of age-related changes were considered in addition to the direct effect of exposure to acrylic acid.

 

In summary, it was shown that 2-year inhalation exposure to acrylic acid caused damaging changes in the respiratory and olfactory epithelium of the nasal cavity, but no progression to tumors was observed. In the 13-week preliminary study, the effects on the olfactory epithelium and respiratory epithelium of the nasal cavity were observed in both males and females with a dose-response relationship. That is, in the 13-week study, atrophy of the olfactory epithelium at 24 ppm or higher group, squamous metaplasia of the respiratory epithelium at 60 ppm or higher group and squamous metaplasia of the olfactory epithelium at 150 ppm or higher group were observed. However, changes attributable to acrylic acid were not observed at 10 ppm group. In the present 2-year study, almost the same biological reactions were observed in the nasal cavity as in the 13-week study. The atrophy of the olfactory epithelium at 40 ppm or higher group and the squamous metaplasia of the respiratory and olfactory epithelium at 160 ppm group were observed, and no changes attributable to the effect of acrylic acid were observed at 10 ppm group. Therefore, it was demonstrated that the lesions in the nasal cavity observed in the 13-week study did not progress even after long-term exposure to acrylic acid.

 

No Observed Adverse Effect Level (NOAEL)

As described above, in the present carcinogenicity study, decreased body weight gain was observed in the treatment groups, and hematology, blood biochemistry, urinalysis and histopathology showed changes probably attributable to acrylic acid.

Obvious changes observed even up to the lowest dose in above changes were atrophy and disarrangement of the olfactory epithelium of the nasal cavity, which were histopathologically observed in both males and females at 40 ppm or higher group. At 10 ppm group, no effect of acrylic acid was observed.

Based on the above results, it was concluded that the no observed adverse effect level (NOAEL) for 2-year inhalation exposure of rats to acrylic acid in the present study was 10 ppm for both males and females based on the atrophy and disarrangement of the olfactory epithelium in the nasal cavity as endpoints.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
478 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
OECD TG 451

Carcinogenicity: via dermal route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 1987 - July 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male and female mice (50 mice/group) of two strains [C3H/HeN Hsd BR] and (Hsd : (ICR)BR), were treated topically with acrylic acid solutions for approx. 21 months.
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Acrylic acid
- Analytical purity: no data
- Source: Aldrich Chemical Company (sample 1), Rohm and Haas Company (sample 2+3)
Species:
mouse
Strain:
other: [C3H/HeN Hsd BR]
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley Inc. (Indianapolis, IN)
- Age at study initiation: approximately 6 1/2 weeks
- Weight at study initiation: 18.64 to 24.65 g for C3H male mice, 15.86 to 21.10 g for C3H female mice
- Housing: one animal/cage section (in divided stainless steel cages)
- Diet (ad libitum): Pelletized Agway Rodent Chow (PROLAB 3000, Agway Inc., Ithaca, NY)
- Water (ad libitum): tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 23.9 °C
- Humidity (%): 40 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

Route of administration:
dermal
Vehicle:
acetone
Details on exposure:
Acrylic acid dosing solutions (1% v/v) were prepared by adding the appropriate amount (ml) of acrylic acid to a volumetric flask and diluting to volume with acetone . The negative control (acetone) was of the same lot used to prepare the test material solutions. Solutions were prepared weekly and stored refrigerated between use.

TEST SITE
- Area of exposure: interscapular region of the back
- Time intervals for shavings or clipplings: on the day prior to application


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 and 100 µL, respectively
- Concentration (if solution): 1 %
- Constant volume or concentration used: yes


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of acrylic acid was measured using gas chromatography (GC). Concentration verification analyses performed on solutions prepared for actual dosing showed analytical values ranging from 92.0 to 111.0 percent of nominal over the 92-week period.
Duration of treatment / exposure:
21 months
Frequency of treatment:
3 times/week
Post exposure period:
In addition, one group of each sex was treated with 25 µL of acrylic acid solution, and a second group was treated with 100 µL of acrylic acid solution 3 days/week for the first 6 weeks of the study only and were then maintained identically (except for the lack of additional dosing) as the animals treated continuously.
Remarks:
Doses / Concentrations:
25 und 100 µL 1% (v/v) acrylic acid solution in acetone (corresponding to approx. 0.26 and 1.04 mg acrylic acid/application; corresponding to approx. 13 and 52 mg/kg bw/application) (estimated mean body weight = 20 g)
Basis:
nominal conc.
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle
Positive control:
Benzo(a)pyrene (0.1 % w/w) in acetone
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (mortality), daily (overt clinical signs)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once during the week

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: once during the week including a detailed recording of non-tumour skin lesions
A proliferative skin lesion in the treatment area was tentatively diagnosed in vivo as a benign tumour (papilloma) when it attained a diameter of
approximately 1 mm. The date of appearance was recorded. Tumours were described. The progress of tumour development was followed and changes in appearance were recorded weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 4 weeks and biweekly thereafter
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

At necropsy, the cranial, thoracic, and abdominal cavities were exposed and examined. The lungs were removed and infused with 10 % neutral buffered formalin. The liver, spleen, kidneys, urinary bladder, mesenteric lymph nodes, and digestive viscera were removed. The removed organs, along with the entire carcass, were immersed in 10 % neutral buffered formalin. If skin tumors were considered to be too large for adequate formalin penetration, they were incised to facilitate proper fixation.
Histopathology was performed on the skin from the application site of all animals and on any nodules, masses or unusual lesions observed at the time of necropsy which were not considered to represent spontaneous lesions common to these strains of mice (e.g. excepting such gross lesions as adrenal nodules). In addition, the liver, spleen, mesenteric lymph nodes, kidneys, urinary bladder, and lungs not deemed to be excessively autolysed were examined histologically for all mice.
Statistics:
Data for body weights were intercompared between each dose group and the respective control group by use of Levene's test for homogeneity of variances, by analysis of variance, and by pooled variance t-tests. The t-tests were used, if the analysis of variance was significant, to delineate which groups differed from the control group. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variances followed, if necessary, by separate variance t-tests. Microscopic lesion frequency data were compared using Fisher's exact tests for two-tailed probability, where appropriate. Mortality between groups within sex was analyzed by life-table analysis. All statistical tests, except the
frequency comparisons were performed using BMDP Statistical Software. The frequency data tests are described in Biometry. The fiducial limit of 0.05
was used as the critical level of significance for all tests.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
No clinical signs of skin irritation at the treatment site or signs of toxicity related to acrylic acid treatment were observed in either sex under any treatment regimen.
Mortality:
no mortality observed
Description (incidence):
Mortality was similar across groups and sexes for approximately the first 18 months of the study. Near the termination of the study, mortality increased in a disproportionate manner with mean survival days being 577, and 608 for the male C3H, and female C3H groups, respectively. To ensure that no groups had less than 50 % survival (as per TSCA guideline 40 CFR Part 798.3300), all treatment groups of a sex were sacrificed when any one treatment group in that sex neared 50% mortality. Based on these criteria, the female C3H animals were sacrificed during Week 92, and the male C3H during Week 89. No differences in mortality were noted between groups treated with acrylic acid and their respective controls.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects on body weight or weight gain were attributed to acrylic acid treatment for either sex at any treatment regimen. A slight trend toward increased mean weight in the BaP treated females in the weeks prior to complete mortality may have resulted from the numbers of tumours in these groups.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No observations were found in the animals from the acrylic acid treated groups that were considered to be related to treatment. Animals from the benzo(a)pyrene treated groups had treatment related lesions which included advanced tumours, papillomas, and enlarged spleens (a common sequela of cutaneous neoplasia in the mouse).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
For the mice sacrificed at the termination of the study, few statistically significant increases in the frequency of microscopic lesions were noted for acrylic acid-treated mice. Those lesions indicated as being significantly increased in one or more acrylic acid-treated groups included:
- (Male C3H):
Ito cell prominence, liver - statistically increased frequency (p< 0.05) for the 6 week 25 µL acrylic acid treated group but not for either the 25 or 100 µL acrylic acid-treated groups treated throughout the study.
Hyperplastic dilated tubules in the kidney - statistically increased frequency for the six week 100 µL acrylic acid treated group (p< 0.05). However, a similar frequency of this microscopic change was present in the 25 µL acetone control group.
- (Female C3H):
Ito cell prominence, liver - statistically increased frequency for the 25 µL (p< 0.05) and 100 µL (p< 0.01) acrylic acid-treated mice.
Mononuclear cell infiltrates, liver - statistically increased frequency for the 25 µL (p< 0.05) but not for the 100 µL acrylic acid-treated groups.
Hepatocellular adenoma, liver - statistically increased frequency (p< 0.05) for the 6 week 25 µL acrylic acid-treated mouse group.
Adnexal atrophy, skin - at statically increased frequency (p< 0.05) for the 100 µL acrylic acid-treated mice.
Lymphoid hyperplasia, mesenteric lymph node - statistically increased frequency (p< 0.05) for the 100 µL acrylic acid treated mice.
Lymphoid hyperplasia, spleen - statistically increased frequency (p< 0.05) for the 100 µL acrylic acid-treated mice.
Tubular pigmentation, kidneys - statistically increased frequency (p< 0.05) for the 6 week 100 µL acrylic acid-treated mice.
With the exception of Ito cell prominence in the liver of female C3H mice, no microscopic findings were increased for both the 25 and 100 µL acrylic acid treated groups. The term "Ito cell prominence" refers to a subjective increase in the numbers and/or size of the Ito cells (the normal fat storing cells) of the liver. This finding is not considered to be of biological significance, and the differences between the acrylic acid-treated groups and their respective control groups are considered to represent random variation.
The increased frequency of adnexal atrophy of the skin in 100 µL acrylic acid treated female C3H mice is also considered to be a apurious occurrence, because there was no evidence of any other effect of acrylic acid on the skin (either clinically or microscopically) within any of the other treatment groups for any sex or strain. Adnexal atrophy is, furthermore, not necessarily an indication of cutaneous toxicity, but may also be indicative of increased grooming behaviour. The increased frequency of lymphoid hyperplasia within the mesenteric lymph nodes of the 100 µL acrylic acid-treated mice is of interest because of the increased frequency of lymphosarcoma also noted for this group.
Nevertheless, the frequencies of the various neoplastic processes for each sex and strain appear to be no different for the acrylic acid-treated mice and for the acetone-treated control mice, with the exception of the frequency of lymphosarcoma (lymphoma) in female C3H mice treated with 100 µL of acrylic acid. Three types of lymphosarcoma were recognized, these being designated as "lymphosarcoma", "composite lymphosarcoma", and "histiocytic lymphosarcoma." Only one of these three diagnosis categories was used for each affected mouse, even though the diagnosis of lymphosarcoma may have been recorded for multiple organs. Seven mice in the 100 µL acrylic acid-treated group and 4 in the 100 µL 6-wk acrylic acid group had lymphosarcoma. One mouse in each of the 25 µL acrylic acid and the 100 µL acetone control had a lymphosarcoma but none were found in mice in the other treatment groups. Because of the increased frequency of lymphosarcoma found within the 100 µL acrylic acid-treated C3H female mouse group, the Sponsor requested a review of the slides from all mice on this study with diagnoses of lymphosarcoma (please refer to "Any other information on results")
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Critical effects observed:
no

The frequency of lymphosarcoma increases sharply in the mouse with advancing age and, for a number of mouse strains evaluated, is seen with a greater frequency in females. Frith reported approximate frequencies (of all lymphosarcoma types combined) of approximately 1% for C3H female mice of between 12 and 18 months of age and approximately 10% for C3H females of greater than 18 months of age. The C3H female 100 µL acrylic acid-treated group on this study had a lymphosarcoma frequency of 14%, slightly higher than the frequency reported for this sex and strain by Frith. The 8% incidence of lymphosarcoma observed in the 100 µL acrylic acid group treated for 6 weeks only was within the historical range reported by Frith. For the other three sexes/strains of mice on this study, there were no statistically significant differences (as determined by Fisher's exact test) in the frequencies of lymphosarcoma between the acrylic acid-treated and acetone-treated control group mice, although numerical increases in the number of lymphomas (in comparison with the control groups) were seen for the 100 µL acrylic acid-treated groups in three of the four sex/strains. No tumours of the treatment area skin were present in any of the acrylic acid-treated or acetone-treated mice.

Results of the Additional Histopathologic Evaluation of Tissues for Hematopoietic Neoplasms:

Non-neoplastic Lesions

Only non-neoplastic lesions related to the hematopoietic system were recorded. These included lymphocytic infiltrations, extramedullary hematopoiesis, increased extramedullary hematopoiesis, plasmacytosis and lymphoid hyperplasia. The only findings of significance were increased extramedullary hematopoiesis in the spleen and extramedullary hematopoiesis in the liver. These findings were increased in Group 7 (Benzo[a]pyrene positive control) and appeared to be related to the increased incidence of skin tumours, since a variety of insults including infections and tumours may result in an increase in extramedullary hematopoiesis.

Neoplastic Lesions

A total of sixteen (16) diagnoses of hematopoietic neoplasms were made in the study. In the present study involving the evaluation of tissues for hematopoietic neoplasms in C3H female mice, only three types of hematopoietic neoplasms were seen. These included histiocytic sarcoma, lymphoblastic lymphoma and follicular center cell lymphoma. Hematopoietic neoplasms are commonly divided into lymphoid and nonlymphoid neoplasms. Histiocytic sarcoma is considered to be a hematopoietic neoplasm developing from the mononuclear phagocyte system and is considered to be a nonlymphoid neoplasm. When tabulations are made concerning the induction of hematopoietic neoplasms by toxic compounds, follicular center cell lymphomas, immunoblastic lymphomas, plasma cell lymphomas and lymphoblastic are generally grouped together since they are derived from lymphocytes. Neoplasms of the mononuclear phagocyte system (histiocytic sarcoma) and myelogenous leukemias (granulocytic, basophilic and erythroleukemia) are generally considered separate from the lymphoid neoplasms.

The two types of lymphoid neoplasms seen in the present study included follicular center cell (FCC) lymphoma and lymphoblastic lymphoma. Lymphoid tumors have been well characterized immunomorphologically in mice and can develop from B lymphocytes, T lymphocytes or non B, non T lymphocytes. Follicular center cell (FCC) lymphomas are derived from B lymphocytes and can usually be identifïed morphologically without the aid of immunocytochemistry. Lymphoblastic lymphomas can develop from either B or T lymphocytes and can not be specifically identified without the aid of immunocytochemistry. Since B or T lymphoblastic lymphomas are commonly grouped with the FCC lymphomas for determining carcinogenicity, immunocytochemistry was not performed in the present study. Although there are no rigid statistical "decision rules" which should be employed in the interpretation of carcinogenicity data, a simple statistical rule was derived by NTP (National Toxicology Program) scientists after a review of 25 bioassays which appears to closely mimic the scientific judgement process used to interpret the results. This "rule" considers as carcinogenic any chemical that produces a high dose increase in a common tumour that is statistically significant at the P< 0.01 level. Common tumours were defined as those with an incidence of a least 1% in the historical controls. In the present study, a slight increase of FCC lymphomas was present in the high dose group (100 µL Acrylic Acid) compared to the other dose groups. This increase was not significant (P= 0.059) when evaluated by the Two Sided Fisher Test. The comparable value for a One Tailed Test is 0.0297. Frith and Wiley reported on the incidence of hematopoietic neoplasms in a number of strains of mice including the C3H strain. Female C3H mice are not commonly used in chronic oncogenicity studies, and there is a lack of historical incidence data for this strain. Frith and Wiley reported an incidence of 9.1 % of lymphoid neoplasms in aged C3H mice. The incidence of hematopoietic neoplasms increases dramatically in female mice between 18 and 24 months of age.

Summary of the histopathologic evaluation:

In the present study no relationship was evident between non-neoplastic lesions of the hematopoietic system and dose of acrylic acid. A small number of hematopoietic neoplasms were present, and a slight increase in follicular center cell lymphoma was evident in the high dose group when compared to the two control groups. The total incidence of lymphomas, particularly follicular center cell lymphomas, was lower in the present study than would normally be expected except in high dose group. Hematopoietic neoplasms, particularly lymphoblastic lymphoma and follicular center cell lymphoma, are commonly seen in most strains of mice including C3H by the time the mice are 18 -24 months of age. It is extremely unusual that Control Group 1 and Control Group 3, each consisting of 50 female C3H mice in the present study, yielded only 1 FCC lymphoma and 1 lymphoblastic lymphoma, respectively. Based primarily on an unusually low incidence of FCC lymphomas compared to historical controls in most groups, it is the authour's opinion that the increase in the high dose group is a spurious finding and is not related to the administration of acrylic acid.

Overall Conclusion:

No clinical signs of skin irritation or toxicity occurred as a result of topical applicationa of 25 µL or 100 µL of 1% acrylic acid to male or female mice of the [C3H/HeN Hsd BR] strain over a 6 -week or up to 21 -month period. In addition, no cutaneous masses occurred in either strain treated with acrylic acid. The frequency of lymphosarcoma was elevated significantly in the female C3H 100 µL acrylic acid-treated group. However, statistically significant increases in lymphosarcoma frequency were not seen in the C3H males or either sex of the ICR mice. The biological significance of these findings is unclear. Body weight was not affected by treatment in either sex of either strain. Topical application of 25 µL of 0.1% benzo(a)pyrene in these strains of mice resulted in an approximate 95% incidence of advanced cutaneous tumours.

Endpoint:
carcinogenicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 1987 - July 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male and female mice (50 mice/group) of two strains [C3H/HeN Hsd BR] and (Hsd : (ICR)BR), were treated topically with acrylic acid solutions for approx. 21 months.
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Acrylic acid
- Analytical purity: no data
- Source: Aldrich Chemical Company (sample 1), Rohm and Haas Company (sample 2+3)
Species:
mouse
Strain:
other: [Hsd : (ICR)BR]
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley Inc. (Indianapolis, IN)
- Age at study initiation: approximately 6 1/2 weeks
- Weight at study initiation: 24.16 to 33.65 g for ICR male mice, 18.55 to 25.87 g for ICR female mice
- Housing: one animal/cage section (in divided stainless steel cages)
- Diet (ad libitum): Pelletized Agway Rodent Chow (PROLAB 3000, Agway Inc., Ithaca, NY)
- Water (ad libitum): tap water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 23.9 °C
- Humidity (%): 40 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

Route of administration:
dermal
Vehicle:
acetone
Details on exposure:
Acrylic acid dosing solutions (1% v/v) were prepared by adding the appropriate amount (ml) of acrylic acid to a volumetric flask and diluting to volume with acetone . The negative control (acetone) was of the same lot used to prepare the test material solutions. Solutions were prepared weekly and stored refrigerated between use.

TEST SITE
- Area of exposure: interscapular region of the back
- Time intervals for shavings or clipplings: on the day prior to application


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 and 100 µL, respectively
- Concentration (if solution): 1 %
- Constant volume or concentration used: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of acrylic acid was measured using gas chromatography (GC). Concentration verification analyses performed on solutions prepared for actual dosing showed analytical values ranging from 92.0 to 111.0 percent of nominal over the 92-week period.
Duration of treatment / exposure:
21 months
Frequency of treatment:
3 times/week
Post exposure period:
In addition, one group of each sex was treated with 25 µL of acrylic acid solution, and a second group was treated with 100 µL of acrylic acid solution 3 days/week for the first 6 weeks of the study only and were then maintained identically (except for the lack of additional dosing) as the animals treated continuously.
Remarks:
Doses / Concentrations:
25 und 100 µL 1% (v/v) acrylic acid solution in acetone (corresponding to approx. 0.26 and 1.04 mg acrylic acid/application; corresponding to approx. 13 and 52 mg/kg bw/application) (estimated mean body weight = 20 g)
Basis:
nominal conc.
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle
Positive control:
Benzo(a)pyrene (0.1 % w/w) in acetone
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (mortality), daily (overt clinical signs)


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once during the week


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: once during the week including a detailed recording of non-tumour skin lesions
A proliferative skin lesion in the treatment area was tentatively diagnosed in vivo as a benign tumour (papilloma) when it attained a diameter of
approximately 1 mm. The date of appearance was recorded. Tumours were described. The progress of tumour development was followed and changes in appearance were recorded weekly.


BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 4 weeks and biweekly thereafter

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

At necropsy, the cranial, thoracic, and abdominal cavities were exposed and examined. The lungs were removed and infused with 10 % neutral buffered formalin. The liver, spleen, kidneys, urinary bladder, mesenteric lymph nodes, and digestive viscera were removed. The removed organs, along with the entire carcass, were immersed in 10 % neutral buffered formalin. If skin tumors were considered to be too large for adequate formalin penetration, they were incised to facilitate proper fixation.
Histopathology was performed on the skin from the application site of all animals and on any nodules, masses or unusual lesions observed at the time of necropsy which were not considered to represent spontaneous lesions common to these strains of mice (e.g. excepting such gross lesions as adrenal nodules). In addition, the liver, spleen, mesenteric lymph nodes, kidneys, urinary bladder, and lungs not deemed to be excessively autolysed were examined histologically for all mice.
Statistics:
Data for body weights were intercompared between each dose group and the respective control group by use of Levene's test for homogeneity of variances, by analysis of variance, and by pooled variance t-tests. The t-tests were used, if the analysis of variance was significant, to delineate which groups differed from the control group. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variances followed, if necessary, by separate variance t-tests. Microscopic lesion frequency data were compared using Fisher's exact tests for two-tailed probability, where appropriate. Mortality between groups within sex was analyzed by life-table analysis. All statistical tests, except the
frequency comparisons were performed using BMDP Statistical Software. The frequency data tests are described in Biometry. The fiducial limit of 0.05
was used as the critical level of significance for all tests.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
No clinical signs of skin irritation at the treatment site or signs of toxicity related to acrylic acid treatment were observed in either sex under any treatment regimen.
Mortality:
no mortality observed
Description (incidence):
Mortality was similar across groups, and sexes for approximately the first 18 months of the study. Near the termination of the study, mortality increased in a disproportionate manner with mean survival days being 554, and 595 for the female ICR, and male ICR groups, respectively. To ensure that no groups had less than 50 % survival (as per TSCA guideline 40 CFR Part 798.3300), all treatment groups of a sex were sacrificed when any one treatment group in that sex neared 50% mortality. Based on these criteria, the female ICR animals were sacrificed during Week 86, and the remaining group sacrificed in Week 92. No differences in mortality were noted between groups treated with acrylic acid and their respective controls.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects on body weight or weight gain were attributed to acrylic acid treatment for either sex at any treatment regimen. A slight trend toward increased mean weight in the BaP treated females in the weeks prior to complete mortality may have resulted from the numbers of tumours in these groups.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No observations were found in the animals from the acrylic acid treated groups that were considered to be related to treatment. Animals from the benzo(a)pyrene treated groups had treatment related lesions which included advanced tumours, papillomas, and enlarged spleens (a common sequela of cutaneous neoplasia in the mouse).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
For the mice sacrificed at the termination of the study, few statistically significant increases in the frequency of microscopic lesions were noted for acrylic acid-treated mice. Those lesions indicated as being significantly increased in one or more acrylic acid-treated groups included:
- (Male ICR):
Hepatocellular degeneration, liver - statistically increased frequency (p< 0.05) for the 25 µL acrylic acid group, but the frequency for the 100 µL acrylic acid group is lower than that for either of the two acetone-treated control groups.
Extramedullary hematopoiesis, spleen - statistically increased frequency (p< 0.01) for the 25 µL acrylic acid group, but the frequency for the 100 µL acrylic acid group is similar to that for both acetone-treated control groups.
Sinus erythrocytosis, mesenteric lymph node - statistically increased frequency (p< 0.05) for the 6 week 100 µL acrylic acid group but not for the 100 µL acrylic acid group treated over the entire course of the study.
- (Female ICR):
Sinus histiocytosis, mesenteric lymph node - statistically increased frequency (p< 0.05) for the 6-Week 100 µl acrylic acid treated group but not for the 100 µL group treated throughout the study.
The increased frequency of hepatocellular adenoma in the 6 week 25 µL acrylic acid-treated female ICR group vs. the respective 25 µL acetone control is considered to represent random variation, because no dose-relationship in the frequency of hepatocellular adenomas is noted. Furthermore, no differences are present in the frequencies of hepatocellular tumours between acrylic acid treated and acetone-treated mice when the data from all of the female ICR mice on study are examined.
Key result
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
> 52 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Critical effects observed:
no

No tumours of the treatment area skin were present in any of the acrylic acid-treated or acetone-treated mice. One 6 week 100 µL acrylic acid-treated male ICR mouse had a squamous cell carcinoma of the subcutis of the left lateral side and left hind leg. This tumour did not involve the overlying skin surface and appeared to possibly have arisen within an epidermal cyst. It was not considered to be related to the acrylic acid treatment.

Conclusion:

No clinical signs of skin irritation or toxicity occurred as a result of topical application of 25 µL or 100 µL of 1% acrylic acid to male or female mice of the [Hsd : (ICR) BR] strain over a 6 -week or up to 21 -month period. In addition, no cutaneous masses occurred in either strain treated with acrylic acid. Body weight was not affected by treatment in either sex. Topical application of 25 µL of 0.1% benzo(a)pyrene in these strains of mice resulted in an approximate 95% incidence of advanced cutaneous tumours.

Endpoint:
carcinogenicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
A group of 40 C3H/HeJ male mice received 25 microliter (µL) applications of acrylic acid as 1.0 % dilutions in acetone. A negative control group received acetone only. The substances were applied to the skin of the back three times weekly for the lifetime of the animals.
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Acrylic acid
- Source: Celanese Chemical Company, Houston, TX
- Analytical purity: approximately 99 % (by weight) (two samples)
Species:
mouse
Strain:
other: C3H/HeJ
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME
- Age at study initiation: 74 to 79 days of age
- Weight at study initiation: test group: 19.3 to 27.9 g, control group: 20.1 to 27.7 g
- Housing: 5/cage
- Diet (ad libitum): Zeigler block feed
- Water (ad libitum): tap water

Route of administration:
dermal
Vehicle:
acetone
Details on exposure:
TEST SITE
- Area of exposure: back of each mouse from which the fur was clipped once weekly.

REMOVAL OF TEST SUBSTANCE
- Washing: no

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples were analyzed for stability as the undiluted liquid using a titration procedure and as a 1 % (by volume) dilution in acetone using a gas chromatographic procedure. In addition, the concentration of acrylic acid in the dilutions (prepared weekly) was measured monthly during the study.
The solutions were shown by these analyses to be quite close to the target concentration of 1 %.
Duration of treatment / exposure:
entire lifetime
Frequency of treatment:
3 days/week
Post exposure period:
no
Remarks:
Doses / Concentrations:
25 µL 1% solution (v/v) in acetone (corresponding to approx. 0.26 mg acrylic acid/application, corresponding to approx. 11 mg/kg bw/application) (mean body weight = 23 g)
Basis:
nominal conc.
No. of animals per sex per dose:
40
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: C3H/HeJ mice were used because of their low incidence of spontaneous skin tumours.
Positive control:
Positive control group consisting of 40 mice received 0.1 % 3-methylcholanthrene (MC).
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION: Yes
- Time schedule for examinations: The mice were carefully examined for lesions of the skin once per month.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Necropsy consisted of a careful examination of the skin and body cavities. All observations were recorded. The skin and any suspect internal tumours from all non-autolyzed mice were fixed in 10 % neutral buffered formalin. Sections were prepared and stained and were examined histologically.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
No signs of skin irritation were observed during the study.
Mortality:
no mortality observed
Description (incidence):
In the group that received 25 µL acrylic acid as 1 % v/v dilutions in acetone, the mean survival time was 515 days, while the mean survival time for the acetone control group was 484 days. The difference was not statistically significant.
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
While one mouse in the acrylic acid group had evidence of epidermal hyperplasia, no neoplasms were found in the skin or subcutis of the acrylic acid-treated mice or in the negative controls.
Key result
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
> 10 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: highest dose tested
Key result
Critical effects observed:
no

No tumors of the skin or subcutis were observed in male mice treated for their lifetime with acrylic acid as a 1.0 % v/v dilution in acetone. In contrast, 39 tumour-bearing rnice were seen in the positive control group. Furthermore, mortality was not affected by treatment. Therefore, acrylic acid was not oncogenic by this testing method.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
52 mg/kg bw/day
Study duration:
chronic
Species:
mouse

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on these data, classification for carcinogencity is not triggerd according to Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.

Additional information

In a valid carcinogenicity study according to OECD TG 451 (BASF AG, 1989; Hellwig et al., 1993) Wistar rats were administered doses of 120, 400 or 1200 ppm (corresponding to approx. 8, 27, and 78 mg/kg bw/d, respectively) acrylic acid (purity: 99 %, stabilized with 200 ppm hydroquinone monomethylether) in drinking water for 26 months (males) or 28 months (females). Except for a slightly reduced water consumption in high-dose males and females no treatment-related clinical, hematological changes were detected in comparison with the controls. The extensive histopathological examination of the preserved tissues revealed that in all three treatment groups, the non-neoplastic tissue changes did not differ from those of the controls. The incidence and organ distribution of tumours found in the groups treated with acrylic acid was not significantly different from those of the controls.

 

In addition, there are several studies which did not fulfill the requirements of guideline testing protocols for regulatory purposes but give valuable additional information on acrylic acid.

 

In a dermal carcinogenicity study no tumours of the skin or subcutis were induced in treated mice or in the vehicle controls (Intercompany Acrylate Study Group, 1982). A group of 40 C3H/HeJ male mice received 25 µL applications of acrylic acid as 1.0 % (v/v) dilutions in acetone. A negative control group received acetone only. The substances were applied to the skin of the back three times a week for lifetime. Histological examination was performed on the dorsal skin of all treated mice and on gross lesions. The mortality rate was not affected by treatment (mean survival time in the acrylic acid group 515 days, in the acetone group 484 days). No signs of skin irritation were observed. While one mouse in the acrylic acid group had evidence of epidermal hyperplasia, no neoplasms were found in the skin or subcutis of the acrylic acid-treated mice or in the negative controls.

 

In another dermal carcinogenicity study 25 or 100 µL of 1 % (v/v) acrylic acid in acetone was administered to two strains of mice (C3H/HeN Hsd BR and Hsd:(ICR)BR) during 21 months (3 times/week). Histopathology was done on the skin, some internal organs and unusual gross lesions. No treatment-related signs of skin irritation, toxicity, clinical signs or skin tumours were observed. There was no treatment-related effect on body weight gain or mortality rate. 7/50 female C3H-mice of the 100 µL acrylic acid treated group revealed a significantly increased frequency of lymphosarcomas compared to the acetone control group (BAMM 1990, 1991). However, statistically significant increases in lymphosarcoma frequency were not seen in the C3H males or either sex of the ICR mice. Lymphosarcomas are commonly seen in most strains of mice which are 18-24 months of age (Frith and Wiley, 1981) and their relation to the treatment was considered to be uncertain.

In a further study the test item was applied dermally on mouse skin 3d/week for 18 months. However, the assessment of findings is not applicable due to missing histological examinations (Celanese 1986). In a vitro study acrylic acid incubated at 37°C for 40 days formed adducts with all four single deoxynucleosides in solution. The relevance of this finding to the potential carcinogenic activity of acrylic acid is questionable given the unusually long incubation interval and the absence of a control incubation of DNA with a non-carcinogenic organic acid of similar structure and molecular weight (Celanese 1984).

Moreover, in a 2-year (104-week) inhalation carcinogenicity study in B6D2F1/Crlj mice and F344/DuCrlCrlj rats, no increase in the incidence of tumors was observed in either sex with exposures up to 32 ppm (mice) and 160 ppm (rats) respectively (JBRC, 2011).

Conclusion

There is no evidence that acrylic acid administered orally to rats or applied dermally to mice is carcinogenic.