N-(1,1-dimethyl-3-oxobutyl)acrylamide

Endpoint summary

Description of key information

Hepatocellular hypertrophy in the liver was recorded in males at 100 and 300 mg/kg and females at 300 mg/kg up to slight severity. At 300 mg/kg, this was accompanied by increased liver weight (up to 23% relative to body weight for both sexes). The morphological change in the liver occurred in the absence of any indicator of hepatocellular toxicity and was therefore not considered adverse. However, the accompanying higher liver weights recorded at 300 mg/kg in both sexes were considered adverse considering the magnitude of increase (i.e. approximately 20% higher than controls).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2015-February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, 1998.

Deviations:

yes

Remarks:

One male at 30 mg/kg (no.17) received on top of the correct dose the high dose of 300 mg/kg on Day26 of the study. ADVIA 2120i was used instead of ADVIA® 120 for the heamatology parameters. The study integrity was not adversely affected by the deviations.

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

EC No 440/2008, B.26 Repeated Dose (90 days) Toxicity (oral), 2008.

Deviations:

yes

Remarks:

One male at 30 mg/kg (no.17) received on top of the correct dose the high dose of 300 mg/kg on Day26 of the study. ADVIA 2120i was used instead of ADVIA® 120 for the heamatology parameters. The study integrity was not adversely affected by the deviations.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

OPPTS 870.3100, EPA 712-C-98-199, 90-Day Oral Toxicity in Rodents, 1998.

Deviations:

yes

Remarks:

One male at 30 mg/kg (no.17) received on top of the correct dose the high dose of 300 mg/kg on Day26 of the study. ADVIA 2120i was used instead of ADVIA® 120 for the heamatology parameters. The study integrity was not adversely affected by the deviations.

Qualifier:

according to guideline

Guideline:

other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2011 by MHLW (0331 No.7), METI (H23.03.29 SeiKyoku No. 5) and MOE (No. 110331009).

Version / remarks:

Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2011 byMHLW (0331 No.7), METI (H23.03.29 SeiKyoku No. 5) and MOE (No. 110331009).

Deviations:

yes

Remarks:

One male at 30 mg/kg (no.17) received on top of the correct dose the high dose of 300 mg/kg on Day26 of the study. ADVIA 2120i was used instead of ADVIA® 120 for the heamatology parameters. The study integrity was not adversely affected by the deviations.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test Item 206895Identification Diacetone acrylamideAppearance White granulateBatch SSDA6B3137Purity/Composition 99.7%Test item storage In refrigerator (2-8°C) protected from lightStable under storage conditions until 31 March 2018 (retest date)Test item handling: use amber glassware or wrap container in aluminium foilpH: 4.6 at concentration 20%

Species:

rat

Strain:

Wistar

Remarks:

Rat: Crl:WI(Han) (outbred, SPF-Quality).

Details on species / strain selection:

Charles River Deutschland, Sulzfeld, Germany.Recognized by international guidelines as the recommended test species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Deutschland, Sulzfeld, Germany- Females nulliparous and non-pregnant: yes- Age at study initiation: approximately 6 weeks- Weight at study initiation: 142-172 g for males and 113-137 g for females- Fasting period before study: no- Housing: groups of 5 animals per sex in Macrolon cages with sterilized sawdust as bedding material andpaper as cage-enrichment- Diet (e.g. ad libitum): free access to pelleted rodent diet (SM R/M-Z from SSNIFF® SpezialdiätenGmbH, Soest, Germany)- Water (e.g. ad libitum): free access to tap water- Acclimation period: at least 5 daysIN-LIFE DATES: From: 24 Nov 2015 To: 24 Feb 2016Acclimatization period At least 5 days before the start of treatment under laboratory conditions.Environmental controls for the animal room are set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for study related activities. Any variations to these conditions were evaluated and maintained in the raw data.Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube.Formulations were placed on a magnetic stirrer during dosing.The dose control system (DCS) was used to verify the dosing procedure.

Vehicle:

propylene glycol

Remarks:

Propylene glycol (Merck, Darmstadt, Germany; from 12 January 2016 onwards Sigma-Aldrich, Steinheim, Germany), specific gravity 1.036.

Details on oral exposure:

Dosing volume: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.Dosing concentrations: 0, 30, 100, 300 mg/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analysed in the formulations of Group 2, 3 and 4 (Week 1, 6 and 13 formulations) were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%), except for Group 3 and 4 formulations in Week 6, that were slightly below the acceptance criteria (mean accuracy of 88.0% and 88.5%, respectively).Based on these results (Week 1, 6 and 13) the concentration of the dosed formulations are considered to be acceptable.No test substance was detected in the Group 1 formulations.The formulations of Group 2 and Group 4 (Week 1, 6 and 13 formulations) were homogeneous (i.e. coefficient of variation ≤ 10%), except for the formulations of Group 2 of Week 6 that were not homogeneous (coefficient of variation of 10.7%).Formulations at the entire range were stable when stored at room temperature protected from light for at least 5 hours (i.e. relative difference ≤ 10%).The long term storage samples were stable at ≤-70°C for 22 days.

Duration of treatment / exposure:

Duration of treatment At least 90 days. Animals were dosed up to the day prior to necropsy.

Frequency of treatment:

Frequency: Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Formulation analyses confirmed that formulations of test item in propylene glycol wereprepared accurately and homogenously, and were stable at room temperature over at least 5 hours.

Dose / conc.:

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

Remarks:

Formulation analyses confirmed that formulations of test item in propylene glycol wereprepared accurately and homogenously, and were stable at room temperature over at least 5 hours.

Dose / conc.:

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

Remarks:

Formulation analyses confirmed that formulations of test item in propylene glycol wereprepared accurately and homogenously, and were stable at room temperature over at least 5 hours.

No. of animals per sex per dose:

10 male, 10 female per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Male and female Wistar (Han) rats, approximately 6 weeks of age on study Day 1, were administered diacetone acrylamide via oral gavage daily for at least 90 consecutive days.group 1: 0 (vehicle) mg/kg bw/day, 10 male, 10 female.group 2: 30 mg/kg bw/day, 10 male, 10 female.group 3: 100 mg/kg bw/day, 10 male, 10 female.group 4: 300 mg/kg bw/day, 10 male, 10 female.- Dose selection rationale: based on dose-range finding study, in which females (3/dose) were exposed to 300 and 500 mg/kg bw/d for 14 days by oral gavage. Results are presented below under any otherinformation.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: twice dailyDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: dailyBODY WEIGHT: Yes- Time schedule for examinations: weeklyFOOD CONSUMPTION:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: YesWATER CONSUMPTION AND COMPOUND INTAKE: Yes- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.OPHTHALMOSCOPIC EXAMINATION: Yes- Time schedule for examinations: all animals at pretest and the control animals and animals at 300 mg/kgbw/d in week 13HAEMATOLOGY: Yes- Time schedule for collection of blood: before necropsy- Anaesthetic used for blood collection: Yes, isoflurane- Animals fasted: Yes, overnight- How many animals: all- Parameters examined were in compliance with OECD 408.CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: before necropsy- Animals fasted: Yes, overnight- How many animals: all- Parameters examined were in compliance with OECD 408 including ALAT, ASAT and ALP, and additionally inorganic phosphate.NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: in week 12 of treatment- Dose groups that were examined: 5/animals/sex/group- Battery of functions tested: sensory activity, grip strength and motor activityMortality / Viability At least twice daily. The time of death was recorded as precisely as possible.IMMUNOLOGY: NoClinical signs At least once daily from start of treatment onwards, detailed clinical observations were made in all animals after dosing (no peak effect of occurrence of clinical signs was observed in the dose range finding study (project 510274)). Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena (collected under Project 510764 for logistic reasons and reported under Project 510272). The time of onset, grade and duration of any observed signs were recorded.Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades are reported, as well as the percentage of animals affected in summary tables.Functional Observations During Week 12 of treatment, the following tests were performed on the first 5 animals/sex/group after dosing at no specific time point, but within a similar time period after dosing for the respective animals (based on the absence of a peak effect in occurrence of clinical signs in the dose range finding study (project 510274)) (abbreviations mentioned in the respective tables indicated between brackets):- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).- fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.Body weights Weekly.Food consumption Weekly.Water consumption Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.OphthalmoscopicExamination (direct) Following instillation of tropicamide solution (5 mg/mL, Minims® Tropicamide 0.5% w/v, Bausch&Lomb Pharma, Brussel, Belgium)both eyes were examined by means of an ophthalmoscope (Heine Beta 200S): at pretest: All animals at Week 13: Groups 1 and 4 animals.Since no treatment-related ophthalmologic findings were noted in Week 13, the eyes of the rats of Groups 2 and 3 were not examined.

Sacrifice and pathology:

On the scheduled day of necropsy, animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination. Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.Samples of the tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands)

Other examinations:

The following organ weights and terminal body weight were recorded from the survivinganimals on the scheduled day of necropsy:Adrenal glands SpleenBrain TestesEpididymides ThymusHeart Uterus (including cervix)Kidneys ProstateLiver Seminal vesicles including coagulating glandsOvaries Thyroid including parathyroidAll organ and tissue samples, as defined under Histopathology (following), were processed, embedded in paraffin wax, cut at a thickness of approximately 3 micrometers and stained with haematoxylin and eosin.The following slides were examined by a pathologist:- all tissues collected at the scheduled sacrifice from all Group 1 and 4 animals,- Liver of all male and female animals and kidneys and adrenal glands of all male animals ofGroups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4,- all gross lesions.All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.Histopathology was subjected to a peer review.

Statistics:

The following statistical methods were used to analyze the data: If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. The Fisher Exact-test (Ref. 3) was applied to frequency data. The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test (Ref. 5) was applied to compare the treated groups to the control group. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant clinical signs during daily clinical observations and no abnormalities during weekly arena observations were noted during the observation period.Salivation noted across the test item treated animals was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item related mortality occurred in this study.One male (animal no. 27, 100 mg/kg) was found dead just prior to necropsy on the scheduled day of necropsy. Since no further deaths occurred in any other groups and no macroscopic or microscopic findings were noted, this death was considered incidental and not related to the test item.No further mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight remained similar to the control level over the study period.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes in clinical biochemistry parameters distinguished treated from control animals:Higher white blood cell count in males at 300 mg/kg.Lower mean corpuscular haemoglobin concentration (MCHC) in females at 100 and 300 mg/kg (not statistically significant at 100 mg/kg).These slight changes in haematology (e.g. white blood cells, and mean corpuscular haemoglobin concentration were considered not adverse, since these changes were slight and no corroborative haematological or morphological findings were noted in this study.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes in clinical biochemistry parameters distinguished treated from control animals:Higher alanine aminotransferase activity (ALAT) in both sexes at 300 mg/kg.Higher total protein in both sexes at 100 and 300 mg/kg (not statistically significant in males at 100 mg/kg).Higher albumin in males at 300 mg/kg.Higher cholesterol in females at 300 mg/kg.Higher calcium in females at 300 mg/kg.These changes in clinical biochemistry parameters were very slight in nature (within the normal range for rats of this age and strain) and considered not toxicologically significant.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At 300 mg/kg, a lower motor activity (total movements and ambulations) was recorded for males and females achieving statistically significance in females only. This was not considered to represent an adverse effect on neurobehaviour, since these results were not supported by clinical observations or other functional observation tests, values were slight in nature (within the normal range for rats of this age and strain), and had no supportive morphological correlates in examined neuronal tissues.No effect on motor activity at 30 and 100 mg/kg was seen. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Enlargement of the liver was present among all test item treated male animals in a dose related distribution (2/10 at 30 mg/kg, 3/10 at 100 mg/kg and 8/10 at 300 mg/kg).The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. There was a significant test item-related increase in liver weight in males and females treated at 300 mg/kg (23% relative to body weight for both sexes) and in kidney weight in females at 100 and 300 mg/kg (10% and 12% relative to body weight resp,) and in males at 300 mg/kg (19% relative to body weight).There were no other test item-related organ weight changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The following test item-related microscopic findings were noted :- Liver: hepatocellular hypertrophy at 100 mg/kg (only males) and 300 mg/kg (males and females)..- Kidneys: increased incidence and/or severity of hyaline droplet accumulation at 100 and 300 mg/kg in males.- Adrenal glands: increased incidence and severity of vacuolation in the zona fasciculata at 300 mg/kg in males.The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hepatocellular hypertrophy in the liver was recorded in males at 100 and 300 mg/kg and females at 300 mg/kg up to slight severity. At 300 mg/kg, this was accompanied by increased liver weight (up to 23% relative to body weight for both sexes).In the male kidney an increased incidence and severity of hyaline droplet accumulation was recorded at 100 and 300 mg/kg up to slight or moderate degree. This finding is considered to likely represent alpha 2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity ofthe tubular epithelium. The occurrence of hyaline droplets is a specific male rat response. This response is not observed in normal female rats, and higher species of either sex, including humans. The hyaline droplets recorded in this study were not accompanied by any indicator of tubular damage, lacked a clear dose response and were therefore not considered an adverse finding. (Alden, CL and Frith CH. Urinary System. In: Handbook of Toxicologic Pathology; Eds. Haschek WM and Rousseaux CG. Academic Press , San Diego. 1991 pp 315-387)In the adrenal glands of males at 300 mg/kg, vacuolation of the zona fasciculata was recorded up to slight degree. Since this lesion was not accompanied by any other indicator of toxicity such as cellular damage this was not considered adverse at these incidences and severities.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Wistar rats were treated with Diacetone acrylamide for 13 weeks by daily oral gavage at dose levels of 30, 100 and 300 mg/kg. The animals in this study showed no toxicologically significant changes in clinical appearance, ophthalmoscopy, body weight and food consumption. A slightly lower motor activity was recorded for both sexes at 300 mg/kg. This was not considered to represent an adverse effect on neurobehaviour, since these results were not supported by clinical observations or other functional observation tests, values were slight in nature (within the normal range for rats of this age and strain), and had no supportive morphological correlates in examined neuronal tissues. Histopathological examination revealed non-adverse morphological changes in liver, kidney and adrenal glands. Hepatocellular hypertrophy in the liver was recorded in males at 100 and 300 mg/kg and females at 300 mg/kg up to slight severity. At 300 mg/kg, this was accompanied by increased liver weight (up to 23% relative to body weight for both sexes). The morphological change in the liver occurred in the absence of any indicator of hepatocellular toxicity and was therefore not considered adverse (Ref 6). However, the accompanying higher liver weights recorded at 300 mg/kg in both sexes were considered adverse considering the magnitude of increase (i.e. approximately 20% higher than controls). Very slight changes were noted in clinical biochemistry parameters (alanine aminotransferase activity, total protein, albumin, cholesterol and calcium). However the changes in clinical biochemistry parameters were very slight in nature (within the normal range for rats of this age and strain) and considered not toxicologically significant. In the male kidney an increased incidence and severity of hyaline droplet accumulation was recorded at 100 and 300 mg/kg up to slight or moderate degree. This finding is considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (Ref 7). A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium. The occurrence of hyaline droplets is a specific male rat response. This response is not observed in normal female rats, and higher species of either sex, including humans. The hyaline droplets recorded in this study were not accompanied by any indicator of tubular damage, lacked a clear doseresponse and were therefore not considered an adverse finding. In the adrenal glands of males at 300 mg/kg, vacuolation of the zona fasciculata was recorded up to slight degree. Since this lesion was not accompanied by any other indicator of toxicity such as cellular damage this was not considered adverse at these incidences and severities. The slight changes in haematology (e.g. white blood cells, and mean corpuscular haemoglobin concentration were considered not adverse, since these changes were slight and no corroborative haematological or morphological findings were noted in this study.

Dose range-finding study: No mortality was observed. Clinical signs included uncoordinated movements and piloerection at 500 mg/kg bw/d and hunched posture and salivation at both dose levels. No effect on body weight and food consumption was noted. No abnormalities at macroscopic examination were seen. Analysis of dose preparations: The concentrations analysed in the formulations of all treated groups (Week 1, 6 and 13 formulations) were in agreement with the target concentrations ( mean accuracies between 90% and 110%, i.e. 91.5 -101.2%), except for formulations at 100 and 300 mg/kg bw/d in Week 6, that were slightly below the acceptance criteria (mean accuracy of 88.0% and 88.5%, respectively). Based on these results (Week 1, 6 and 13) the concentration of the dosed formulations are considered to be acceptable. No test substance was detected in the Group 1 formulations. The formulations at 30 and 300 mg/kg bw/d (Week 1, 6 and 13 formulations) were homogeneous (coefficient of variation ≤ 10%, i.e. 0.80 -2.9%), except for the formulations at 30 mg/kg bw/d of Week 6 that were not homogeneous (coefficient of variation of 10.7%). Since this was only a slight deviation and homogeneity was confirmed in Week 1 and 13, this was considered not to affect the nominal dose level during the 90- day exposure. Formulations at the entire range were stable when stored at room temperature protected from light for at least 5 hours (1.1 -1.2%, i.e. relative difference ≤ 10%). The long term storage samples were stable at ≤-70°C for 22 days.

Conclusions:

A NOAEL of 100 mg/kg was established based on the higher liver weight in males and females at 300 mg/kg.

Executive summary:

Repeated dose 90-day oral toxicity study with Diacetone Acrylamide by daily gavage in the rat.

Based on the results of a 14 -day range finding study, the dose levels for this 90-day oral gavage study were selected to be 0, 30, 100 and 300 mg/kg.

The test item, formulated in propylene glycol, was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Evaluated parameters Chemical analyses of formulations were conducted during the study to assess accuracy, homogeneity and stability at room temperature over 5 hours. The following parameters were evaluated: clinical signs daily; functional observation tests in Week 12; body weight and food consumption weekly; ophthalmoscopy at pretest and in Week 13; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Results:

Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously, and were stable at room temperature over at least 5 hours.

Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously, and were stable at room temperature over at least 5 hours.

The animals in this study showed no toxicologically significant changes in clinical appearance, ophthalmoscopy, body weight and food consumption.

A slightly lower motor activity was recorded for both sexes at 300 mg/kg. This was not considered to represent an adverse effect on neurobehaviour, since these results were not supported by clinical observations or other functional observation tests, values were slight in nature (within the normal range for rats of this age and strain), and had no supportive morphological correlates in examined neuronal tissues.

Histopathological examination revealed non-adverse morphological changes in liver, kidney and adrenal glands. Hepatocellular hypertrophy in the liver was recorded in males at 100 and 300 mg/kg and females at 300 mg/kg up to slight severity. At 300 mg/kg, this was accompanied by increased liver weight (up to 23% relative to body weight for both sexes). The morphological change in the liver occurred in the absence of any indicator of hepatocellular toxicity and was therefore not considered adverse. However, the accompanying higher liver weights recorded at 300 mg/kg in both sexes were considered adverse considering the magnitude of increase (i.e. approximately 20% higher than controls). Very slight changes were noted in clinical biochemistry parameters (alanine aminotransferase activity, total protein, albumin, cholesterol and calcium). However the changes in clinical biochemistry parameters were very slight in nature (within the normal range for rats of this age and strain) and considered not toxicologically significant. In the male kidney an increased incidence and severity of hyaline droplet accumulation was recorded at 100 and 300 mg/kg up to slight or moderate degree. This finding is considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium. The occurrence of hyaline droplets is a specific male rat response. This response is not observed in normal female rats, and higher species of either sex, including humans. The hyaline droplets recorded in this study were not accompanied by any indicator of tubular damage, lacked a clear doseresponse and were therefore not considered an adverse finding. In the adrenal glands of males at 300 mg/kg, vacuolation of the zona fasciculata was recorded up to slight degree. Since this lesion was not accompanied by any other indicator of toxicity such as cellular damage this was not considered adverse at these incidences and severities.

The slight changes in haematology (e.g. white blood cells, and mean corpuscular haemoglobin concentration were considered not adverse, since these changes were slight and no corroborative haematological or morphological findings were noted in this study. In conclusion, a NOAEL of 100 mg/kg was established based on the higher liver weight in males and females at 300 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Guideline study (90 day rats; key study) and 2 subchronic neurotoxicity test with rats and 1 with mice were evaluated.The key guideline study describes a NOAEL of 100 mg/kg bw/day.

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Hepatocellular hypertrophy in the liver was recorded in males at 100 and 300 mg/kg and females at 300 mg/kg up to slight severity. At 300 mg/kg, this was accompanied by increased liver weight (up to 23% relative to body weight for both sexes). The morphological change in the liver occurred in the absence of any indicator of hepatocellular toxicity and was therefore not considered adverse. However, the accompanying higher liver weights recorded at 300 mg/kg in both sexes were considered adverse considering the magnitude of increase (i.e. approximately 20% higher than controls).
A decreased body weight was observed in 2 subchronic neurotoxicity studies with rats at high doses of 29 000 mg/L drinking water, corresponding to ca. 3000 mg/kg bw/d. Today there are no indications of further toxic effects of the submitted substance after repeated dosing.
A supporting subchronic neurotoxicity study with mice reports a NOEL of 651 mg/kg bw/d.
As only 2 LOELs of ca. 3000 mg/kg bw/d are available for the usually preferred species rat, the lower NOEL for mice is used to preliminarily calculate the DNELs.


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Today there are no indications of a toxic effect of the submitted substance after repeated dosing. A waiver is proposed for not testing with the inhalation route.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Today there are no indications of a toxic effect of the submitted substance after repeated dosing. A waiver is proposed for not testing with the inhalation route.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Today there are no indications of a toxic effect of the submitted substance after repeated dosing. A waiver is proposed for not testing with the inhalation route.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Today there are no indications of a toxic effect of the submitted substance after repeated dosing. A waiver is proposed for not testing with the inhalation route.

Justification for classification or non-classification

Today there are no indications of a relevant toxic effect of the submitted substance after repeated dosing according to the criteria of Regulation 1272/2008 CLP. No classification is required.