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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects on fertility have been identified.

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction

Remarks:

other: Effects on reproductive organs

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Publication in a recognized journal.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Comparison of the known neurotoxicity and the effects on testis for acrylamide and possible effects for 13 related compounds.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

other: ddY

Sex:

male

Details on test animals or test system and environmental conditions:

- Age at study initiation: 5-6 weeks- Weight at study initiation: 29 +- 2.2 g- Housing: 5-7 per cage- Diet: ad libitum- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

physiological saline

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

8 - 10 weeks

Frequency of treatment:

twice weekly

Remarks:

Doses / Concentrations:Doses ranging from 1/2 to 1/5 of the LD50. The LD50 = 7.7. mmol/kg. Doses therefore ranging from 261 to 651 mg/kg bw.Basis:

No. of animals per sex per dose:

5-7 animals per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

To examine the effect of metabolic activation, sodium phenobarbital (PB), which was prepared from phenobarbital before use, was given intraperitoneally at 50 mg/kg for five successive days per week, from one week before, up until the last week of treatment with the test compounds

Postmortem examinations (offspring):

Histopathological study of the testis and examination of blood: After treatment with the test compounds for 8-10 weeks, mice were killed under ether anesthesia for histology and blood examination. The testis was weighed and fixed in 10% neutral formalin, processed, and embedded in paraffin. Ten-micron sections were stained with hematoxylin and eosin. Blood was taken from the right atrium with a heparinized syringe. Measurements of red and white blood cell counts, hemoglobin concentration, and hematocrit value, and differentiation of white blood cells, were conducted by routine methods.

Statistics:

Intergroup comparison was conducted by the Student's t-test.

Clinical signs:

not examined

Body weight and weight changes:

no effects observed

Description (incidence and severity):

body weight

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

body weight

Organ weight findings including organ / body weight ratios:

not examined

Other effects:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Effect on testis: Atrophy and reduced testis weight was noted in 5 compounds, but not in diacetone acrylamide. No histopathological lesions were reported for diacetone acrylamide.Effect of PB treatment: diacetone acrylamide was not tested.Blood study: Effects of test compounds on the blood were examined after the last treatment. Only one compound, N,N'-methylene-bis-acrylamide, produced marked effects on red and white blood cell counts, hemoglobin concentration and hematocrit value.

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Reproductive effects observed:

not specified

Conclusions:

Diacetone acrylamide did not produce lesions of the testes in this type of experiment.

Executive summary:

Neurotoxicity of acrylamide and related compounds and their effects on the testis after repeated oral doses were studied in mice. Of fourteen analogues tested, five produced neuropathy; but diacetone acrylamide did not.

No effects on testis or blood were noted for diacetone acrylamide.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Additional information

Justification for selection of Effect on fertility:
There are no indications of an effect of the submitted substance on the reproduction.

Effects on developmental toxicity

Description of key information

Developmental toxicity:

An increase of slightly to moderately malaligned sternebrae at the high dose level (300 mg/kg bw/day) was reported.

Maternal toxicity:

Body weight gain corrected for weight of the uterus showed a statistically significant dose related decrease from 30 mg/kg onwards. For the control, low, mid and high dose groups, corrected body weight gain was 26.5, 20.0, 16.8 and 14.0 grams, respectively. At 30 and 100 mg/kg no concurrent findings were noted for body weight, food consumption, clinical signs and macroscopic examination. Furthermore, although slightly reduced these females still gained weight and the values were within the range of the historical control data. Therefore, the reduced body weight gain corrected for weight of the uterus at 30 and 100 mg/kg was considered of no toxicological relevance. However, the reduced corrected body weight gain at 300 mg/kg was accompanied by a reduced body weight at Day 21 postcoitum, and reduced body weight gain and food consumption. Furthermore, the value of 14.0 grams for the 300 mg/kg treated females was below the fifth percentile of the historical control data. Taken together, this finding at 300 mg/kg was considered to be adverse.

In conclusion, based on the results in this prenatal developmental toxicity study the following No Observed Adverse Effect Levels (NOAEL) for Diacetone acrylamide were established:

NOAEL maternal toxicity: 100 mg/kg bw/day

NOAEL developmental toxicity: 100 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2016 - May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Organization of Economic Co-operation and Development Guidelines (OECD) for testingof Chemicals Guideline 414, Prenatal Developmental Toxicity Study, January 2001.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.31: "Prenatal Developmental Toxicity Study".Official Journal of the European Union No. L142, May 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

The United States Environmental Protection Agency (EPA) Health Effects Test GuidelinesOPPTS 870.3700, Prenatal Developmental Toxicity Study, August 1998.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test item 206895/AIdentification 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

Species:

rat

Strain:

Wistar

Remarks:

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

Details on test animals or test system and environmental conditions:

Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).Environmental controls for the animal room were 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. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.Diet, water, bedding and cage-enrichment/nesting material 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

Vehicle:

propylene glycol

Details on exposure:

Oral gavage, using a plastic feeding tube.Formulations were placed on a magnetic stirrer during dosing.5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No test item was detected in the Group 1 formulations.The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

ScheduleMating at Supplier 18 to 21 April 2016Delivery 19 and 21 April 2016Start treatment 24 April 2016Necropsy 09 to 12 May 2016Experimental completion date 12 May 2016 (end of in-life phase)

Duration of treatment / exposure:

From Days 6 to 20 post-coitum, inclusive.

Frequency of treatment:

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

Duration of test:

20 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

22 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Experimental starting date 12 February 2016 (first delivery of mated females of the dose range finding study)Mating at Supplier 18 to 21 April 2016Delivery 19 and 21 April 2016Start treatment 24 April 2016Necropsy 09 to 12 May 2016Experimental completion date 12 May 2016 (end of in-life phase)

Maternal examinations:

All animals were sacrificed on Day 21 post-coitum using an oxygen/carbon dioxide procedure and subsequently subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).CAGE SIDE OBSERVATIONS: Yes- Time schedule: twice dailyDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: once dailyBODY WEIGHT: Yes- Time schedule for examinations: days 2, 6, 9, 12, 15, 18 and 21 post-coitumFOOD CONSUMPTION: Yes- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitumWATER CONSUMPTION: Yes- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspectedPOST-MORTEM EXAMINATIONS: Yes- Sacrifice on gestation day 21- Organs examined: external, thoracic and abdominal examination with special attention being paid to the reproductive organs

Ovaries and uterine content:

Each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine:- The number of corpora lutea.- The weight of the (gravid) uterus.- The number and distribution of live and dead fetuses.- The number and distribution of embryo-fetal deaths (early and late resorptions).- The weight of each fetus.- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).- Externally visible macroscopic fetal abnormalities.In case implantations were not macroscopically visible, the uterus was stained using the Salewski technique in order to determine any former implantation sites (Salewski staining prepared at Charles River Laboratories Den Bosch using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Fetal examinations:

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).External:Each viable fetus was examined in detail, weighed and sexed. All live fetuses were euthanized by administration of approximately 0.05 mL (=10mg) of sodium pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater) into the oral cavity using a small flexible plastic or metal feeding tube.Visceral (Internal):Approximately one-half of the fetuses in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (Ref. 2).This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar (Ref. 3). The sex of all fetuses was confirmed by internal examination.The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution (Klinipath, Duiven, The Netherlands) for soft-tissue examination of all groups using the Wilson sectioning technique (Ref. 4). After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% buffered formalin.Any remaining tissues (from the fetuses used for fresh visceral examination) were discarded.The carcasses were processed and stained with Alizarin Red S (as described below), but not examined.Skeletal:From the other one-half of the fetuses in each litter (all groups), the sex was confirmed by internal examination. All fetuses were eviscerated, fixed in 96% aqueous ethanol, macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson (Ref. 5). Skeletal examination was done for one-half of the fetuses (i.e. the fetuses withheads).The specimens were archived in glycerin (BRENNTAG Nederland B.V., Dordrecht, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

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. 6) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.- The Steel-test (Ref. 7 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.- The Fisher Exact-test (Ref. 8) was applied to frequency data.- The Mann Whitney test (Ref. 9) was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 10) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test (Ref. 11) was used to compare the compound-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 might be rounded off before printing. Therefore, two groups might display thesame printed means for a given parameter, yet display different test statistics values.No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

For each litter the following calculations were performed:Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) number of corpora lutea x 100Post-implantation loss (%) = (number of imnupmlanbteart ioofn i msiptelsa n-tnatuimonb seirt eosf live fetuses) x 100The fetal developmental findings were summarized by: 1) presenting the incidence of a givenfinding both as the number of fetuses and the number of litters available for examination inthe group; and 2) considering the litter as the basic unit for comparison, calculating thenumber of affected fetuses as a mean litter proportion on a total group basis, where:Viable fetuses affected/litter (%) = numnbuerm obfevr ioafblvei afbelteu sfeest uasfefse/cltiettde/rlitter x 100

Historical control data:

Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)Gestation Day 21Study Date Range: 2014 - 2015

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signs were noted up to 300 mg/kg.The single occurrence of alopecia (female no. 48) remained within the range of background findings to be expected for rats of this strain and age.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in this study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall, a trend towards lower body weight gain was observed in all treatment groups. At 300 mg/kg body weight gain was significantly reduced compared to the control group from Day 9 post-coitum onwards, which resulted in 7% lower body weights on Day 21 post-coitumat this dose level. A very slight, non-significant, reduction in body weight gain was noted at 30 and 100 mg/kg.Body weight gain corrected for weight of the uterus showed a statistically significant dose related decrease from 30 mg/kg onwards. For the low, mid and high dose groups, corrected body weight gain was respectively 20.0, 16.8 and 14.0 grams, compared to 26.5 grams in thevehicle control group. These corrected body weights in the low and mid dose groups were within the range of the historical control data, whereas the 14.0 grams for the 300 mg/kg treated females was below the fifth percentile.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In all treatment groups, a trend towards reduced food consumption was observed in the first week of treatment. In females treated at 100 and 300 mg/kg, absolute and relative food consumption were statistically significantly reduced between Days 6 and 15 post-coitum. At 30 mg/kg the reduction in food consumption was only slight and did not reach a level of statistical significance.

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

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related macroscopic findings were noted up to 300 mg/kg.The diaphragmatic hernia of the liver observed in female no. 80 (300 mg/kg group) and alopecia in the chest region for female no. 21 (control group) were considered to be incidental findings.

Number of abortions:

no effects observed

Description (incidence and severity):

There were no treatment related effects on litter size for any group.Mean litter sizes were 10.4, 10.5, 11.2 and 9.8 for the control, 30, 100 and 300 mg/kg groups, respectively.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no treatment related effects on litter size for any group.Mean litter sizes were 10.4, 10.5, 11.2 and 9.8 for the control, 30, 100 and 300 mg/kg groups, respectively.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no treatment related effects on litter size for any group.Mean litter sizes were 10.4, 10.5, 11.2 and 9.8 for the control, 30, 100 and 300 mg/kg groups, respectively.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological examination were 228 (22), 231 (22), 236 (21) and 216 (22) in Groups 1, 2, 3, and 4, respectively. External examination was done for all fetuses, visceral examination was done for approximately half of the fetuses of all groups, and skeletal examination was done for the other half of fetuses. Females A086 and A088 had one viable fetus only, which were selected for visceral examination. Consequently, 20 litters were available for skeletal evaluation in Group 4. For fetus A087-11 that wasassigned to a visceral examination, the fresh visceral examination was not continued by the soft tissue cephalic examination due to presence.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No treatment related macroscopic findings were noted up to 300 mg/kg.The diaphragmatic hernia of the liver observed in female no. 80 (300 mg/kg group) andalopecia in the chest region for female no. 21 (control group) were considered to be incidentalfindings.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females, except for one 100 mg/kg treated female (no. 52), were pregnant with viable fetuses.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: maternal body weight

Description (incidence and severity):

In all treatment groups, a trend towards reduced body weight gain and concurrent reduced food consumption was observed. At 30 and 100 mg/kg these effects were only slight and only statistically significant for food consumption at 100 mg/kg and were therefore considered not to be adverse. However, for the 300 mg/kg treated females body weight gain, body weight at Day 21 post-coitum and food consumption between Days 6 and 15 post-coitum were significantly reduced. As these effects were more pronounced these were considered to be signs of maternal toxicity at this dose level.Body weight gain corrected for weight of the uterus showed a statistically significant dose related decrease from 30 mg/kg onwards. For the control, low, mid and high dose groups, corrected body weight gain was 26.5, 20.0, 16.8 and 14.0 grams, respectively. At 30 and 100 mg/kg no concurrent findings were noted for body weight, food consumption, clinical signs and macroscopic examination. Furthermore, although slightly reduced these females still gained weight and the values were within the range of the historical control data.Therefore, the reduced body weight gain corrected for weight of the uterus at 30 and100 mg/kg was considered of no toxicological relevance. However, the reduced corrected body weight gain at 300 mg/kg was accompanied by a reduced body weight at Day 21 postcoitum, and reduced body weight gain and food consumption. Furthermore, the value of 14.0 grams for the 300 mg/kg treated females was below the fifth percentile of the historical control data. Taken together, this finding at 300 mg/kg was considered to be adverse.

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 100 and 300 mg/kg combined fetal body weights were reduced to 4.9 grams in both treated groups compared to 5.1 grams in the vehicle control group. This was only statistically significant for the 100 mg/kg group, due to larger variation at 300 mg/kg. This larger variation could be explained by the high mean litter weights for two females, which each had one viable fetus only, which was relatively large.This could partly be explained by the effect on maternal food consumption during the first week of treatment in these groups. Skeletal examination did not show indications of growth retardation and therefore, the effect on fetal body weights at 100 and 300 mg/kg was considered not adverse.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Mean litter sizes were 10.4, 10.5, 11.2 and 9.8 for the control, 30, 100 and 300 mg/kg groups, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio: The male/female ratio was unaffected by treatment up to 300 mg/kg.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological examination were 228 (22), 231 (22), 236 (21) and 216 (22) in Groups 1, 2, 3, and 4, respectively. External examination was done for all fetuses, visceral examination was done for approximately half of the fetuses of all groups, and skeletal examination was done for the other half of fetuses. Females A086 and A088 had one viable fetus only, which were selected for visceral examination. Consequently, 20 litters were available for skeletal evaluation in Group 4. For fetus A087-11 that was assigned to a visceral examination, the fresh visceral examination was not continued by the soft tissue cephalic examination due to presence of external head malformations. Instead, this fetus was examined skeletally as well.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to 300 mg/kg.Two externally malformed fetuses were observed in this study; both in the high dose group.Fetus A074-04 had a small lower jaw and in fetus A087-11 the same finding was seen together with meningoencephalocele. In the historical control data, a small or absent lower jaw (skeletally) was previously noted, as was the exencepahly, which is related to meningoencephalecele. The incidence of a small lower jaw was 0.7% per litter at 300 mg/kg, whereas the historical control maximum value is 0.8% per litter. Therefore, the occurrence of two malformed fetuses at the high dose were considered a chance finding.External variations were not seen in any of the groups.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The variation of slightly to moderately malaligned sternebrae was statistically significantly increased in the high dose group. The incidences of this finding were 19.9%, 25.7%, 28.2% and 41.4% per litter in 0, 30, 100 and 300 mg/kg treatment groups, respectively. This also resulted in an increased incidence of total variations in the high dose group that was statistically significant as well (93.5% versus control value 80.9% per litter). At 300 mg/kg, the incidence of the slightly to moderately malaligned sternebrae was far above the historical control maximum value (41.4% versus 21.3% per litter). It should be noted that the background as observed in the concurrent control (19.9% per litter) was close to that limit.The incidences of this variation in the low and mid dose groups (25.7% and 28.2% per litter, respectively) were also above the historical upper limit. However, because no clear dose response was noted; statistical significance could not be established for the 30 and 100 mg/kg groups; and the percentages at 30 and 100 mg/kg were only slightly higher compared to the concurrent control, only the increase at the highest dose level was considered to be treatment related.Additionally, the variation of 14th full ribs was noted at a mean litter incidence of 9.4%, 9.3%, 14.3% and 23.7% per litter in the control, 30, 100 and 300 mg/kg Groups, respectively.The incidences in the mid and high dose groups were not statistically significantly increased, but were higher than the historical control maximum value, i.e. 13.1% per litter. The mean litter incidence at 100 mg/kg (14.3% per litter) was only slightly above the maximum value in the historical control data and the incidence in the concurrent control group was relatively high compared to the historical control mean incidence (9.4% versus 5.7% per litter, respectively). Taken together, the increased incidence at 100 mg/kg was considered to be of no toxicological relevance. However, the incidence of 14th full ribs at 300 mg/kg was more than twice as high as the vehicle control group (23.7% versus 9.4% per litter, respectively) and therefore, this finding was considered adverse related at this dose level.Other skeletal variations that were noted occurred in the absence of a dose-related incidence trend or occurred infrequently. Therefore, these findings were not considered to be treatment related.Only two different malformations were revealed at skeletal examination. Bent limb bones (scapulas and/or humeri) were observed in two controls (A001-09 and A013-07), in one Group 2 fetus (A041-10) and in one Group 4 fetus (A075-05). One Group 2 fetus A031-12 had a vertebral centra anomaly. The group distribution and low incidence of these malformations do not suggest any treatment related effect.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to 300 mg/kg.The only viscerally malformed fetus, A076-01 at 300 mg/kg, had a cardiovascular malformation for which an interrupted aortic arch was observed together with a ventricular septum defect. As this was a single occurrence, it was considered a chance finding.The visceral variations that were noted were small supernumerary lobe(s) and discolored liver, partially undescended thymus horns and convoluted ureters. These variations occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that werewithin the range of available historical control data. The absence of the finding of an appendix of the liver in the high dose group (statistically significant compared to vehicle controls) was considered to be of no toxicological relevance.

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:

other: skeletal variations, i.e. malaligned sternebrae and 14th rib

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:

other: skeletal variations, i.e. malaligned sternebrae and 14th rib

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: sternum

skeletal: rib

Description (incidence and severity):

At 300 mg/kg, the incidence of slightly to moderately malaligned sternebrae was significantly increased compared to the control group (41.4% versus 19.9% per litter) and was also far above the historical control maximum value (21.3% per litter), therefore the increase of slightly to moderately malaligned sternebrae at the high dose level was considered to be treatment related.The mean litter incidence of 14th full ribs was more than twice as high compared to thevehicle control group (23.7% versus 9.4% per litter, respectively) and therefore, this increased incidence of 14th full ribs was considered adverse at this dose level.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Mated female Wistar Han rats were assigned to four dose groups, each containing twenty-two animals. The test item was administered once daily by gavage from Day 6 to 20 post-coitum at doses of 30, 100 and 300 mg/kg (Groups 2, 3 and 4 respectively). The rats of the control group (Group 1) received the vehicle, propylene glycol, alone. Accuracy and homogeneity of formulations were demonstrated by analyses.

Maternal findings

In all treatment groups, a trend towards reduced body weight gain and concurrent reduced food consumption was observed. At 30 and 100 mg/kg these effects were only slight and only statistically significant for food consumption at 100 mg/kg and were therefore considered not to be adverse. However, for the 300 mg/kg treated females body weight gain, body weight at Day 21 post-coitum and food consumption between Days 6 and 15 post-coitum were significantly reduced. As these effects were more pronounced these were considered to be signs of maternal toxicity at this dose level. Body weight gain corrected for weight of the uterus showed a statistically significant dose related decrease from 30 mg/kg onwards. For the control, low, mid and high dose groups, corrected body weight gain was 26.5, 20.0, 16.8 and 14.0 grams, respectively. At 30 and 100 mg/kg no concurrent findings were noted for body weight, food consumption, clinical signs and macroscopic examination. Furthermore, although slightly reduced these females still gained weight and the values were within the range of the historical control data. Therefore, the reduced body weight gain corrected for weight of the uterus at 30 and 100 mg/kg was considered of no toxicological relevance. However, the reduced corrected body weight gain at 300 mg/kg was accompanied by a reduced body weight at Day 21 postcoitum, and reduced body weight gain and food consumption. Furthermore, the value of 14.0 grams for the 300 mg/kg treated females was below the fifth percentile of the historical control data. Taken together, this finding at 300 mg/kg was considered to be adverse.

Developmental findings

At 300 mg/kg, the incidence of slightly to moderately malaligned sternebrae was significantly increased compared to the control group (41.4% versus 19.9% per litter) and was also far above the historical control maximum value (21.3% per litter), therefore the increase of slightly to moderately malaligned sternebrae at the high dose level was considered to be treatment related. The mean litter incidence of 14th full ribs was more than twice as high compared to the vehicle control group (23.7% versus 9.4% per litter, respectively) and therefore, this increased incidence of 14th full ribs was considered adverse at this dose level. Fetal body weights were reduced at 100 and 300 mg/kg, which could partly be explained by the effect on maternal food consumption during the first week of treatment in these groups. Skeletal examination did not show indications of growth retardation and therefore, the effect on fetal body weights at 100 and 300 mg/kg was considered not adverse. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Diacetone acrylamide was established as being 100 mg/kg.

Results dose range finding study: Maternal findings One female treated at 500 mg/kg showed body weight loss between Days 6 and 12 post-coitum and reduced body weight gain thereafter. When this female was found lethargic and showing muscle twitching and flat posture on Day 20 post-coitum, this female was sacrificed for animal welfare reasons. At necropsy, an enlarged heart was noted for this animal. No other mortality occurred in this study. No treatment related clinical signs were noted up to and including 500 mg/kg, except for this female. Reduced body weight gain was observed in the 300 and 500 mg/kg treated females, which was statistically significant at 500 mg/kg. Body weight gain corrected for weight of the uterus was significantly reduced in females treated at 300 mg/kg, a similar trend was observed at 100 mg/kg. A corrected body weight loss was observed in 500 mg/kg treated animals. During the treatment period, food consumption was reduced in 500 mg/kg treated females, reaching statistical significance on Days 6-12 and Days 18-21 post-coitum. A similar trend was observed in the 300 mg/kg group in the first week of treatment. Absolute food consumption was statistically significantly reduced between Days 18-21 post-coitum in the 100 mg/kg group. Up to and including 500 mg/ kg, no treatment related abnormalities were noted at macroscopic examination.

Fetal findings

Litter sizes were within normal limits for all groups. The male:female ratios were equal in litters of all groups. Fetal body weights were decreased for male and female fetuses in the 500 mg/kg dose group. The incidence of early resorptions and consequently the incidence of post-implantation loss was significantly increased at 500 mg/kg compared to the vehicle controls (7.0 versus 0.0% per litter, respectively). No external abnormalities were noted for the fetuses in any of the groups.

Conclusions:

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Diacetone acrylamide was established as being 100 mg/kg bw/day.

Executive summary:

A prenatal developmental toxicity study of Diacetone acrylamide in rats by oral gavage.

Guidelines

The study procedures described in this report were based on the following guidelines:

1) Organization of Economic Co-operation and Development Guidelines (OECD) for testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, January 2001.

2) Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.31: "Prenatal Developmental Toxicity Study". Official Journal of the European Union No. L142, May 2008.

3) The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study, August 1998.

Rationale for dose levels Dose levels were based on a dose range finding study. Four groups of 6 females were exposed to 0, 100, 300 and 500 mg/kg for Days 6 to 20 post-coitum inclusive by oral gavage. One female treated at 500 mg/kg was euthanized at Day 20 post-coitum when found lethargic, with flat posture and exhibiting muscle twitching. In combination with a corrected body weight loss at 500 mg/kg, 300 mg/kg was selected as the high dose in the main study. Study outline Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 20 post-coitum at doses of 0, 100 and 300 mg/kg (Groups 2, 3 and 4 respectively). The rats of the control group received the vehicle, propylene glycol, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy and homogeneity. On Day 21 post-coitum, all animals were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. Gross lesions were collected and fixed from all animals at necropsy. A laparohysterectomy was performed on each female. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses was decapitated and the heads were fixed in Bouin’s fixative; these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses was processed and stained with Alizarin Red S for skeletal examinations.

RESULTS

Accuracy and homogeneity of formulations were demonstrated by analyses. Maternal findings In all treatment groups, a trend towards reduced body weight gain and concurrent reduced food consumption was observed. At 30 and 100 mg/kg these effects were only slight and only statistically significant for food consumption at 100 mg/kg and were therefore considered not to be adverse. However, for the 300 mg/kg treated females body weight gain, body weight at Day 21 post-coitum and food consumption between Days 6 and 15 post-coitum were significantly reduced. As these effects were more pronounced these were considered to be signs of maternal toxicity at this dose level. Body weight gain corrected for weight of the uterus showed a statistically significant dose related decrease from 30 mg/kg onwards. For the control, low, mid and high dose groups, corrected body weight gain was 26.5, 20.0, 16.8 and 14.0 grams, respectively. At 30 and 100 mg/kg no concurrent findings were noted for body weight, food consumption, clinical signs and macroscopic examination. Furthermore, although slightly reduced these females still gained weight and the values were within the range of the historical control data. Therefore, the reduced body weight gain corrected for weight of the uterus at 30 and 100 mg/kg was considered of no toxicological relevance. However, the reduced corrected body weight gain at 300 mg/kg was accompanied by a reduced body weight at Day 21 postcoitum, and reduced body weight gain and food consumption. Furthermore, the value of 14.0 grams for the 300 mg/kg treated females was below the fifth percentile of the historical control data. Taken together, this finding at 300 mg/kg was considered to be adverse. Developmental findings At 300 mg/kg, the incidence of slightly to moderately malaligned sternebrae was significantly increased compared to the control group (41.4% versus 19.9% per litter) and was also far above the historical control maximum value (21.3% per litter), therefore the increase of slightly to moderately malaligned sternebrae at the high dose level was considered to be treatment related. The mean litter incidence of 14th full ribs was more than twice as high compared to the vehicle control group (23.7% versus 9.4% per litter, respectively) and therefore, this increased incidence of 14th full ribs was considered adverse at this dose level. Fetal body weights were reduced at 100 and 300 mg/kg, which could partly be explained by the effect on maternal food consumption during the first week of treatment in these groups. Skeletal examination did not show indications of growth retardation and therefore, the effect on fetal body weights at 100 and 300 mg/kg was considered not adverse.

CONCLUSION

Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Diacetone acrylamide was established as being 100 mg/kg.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

guideline study

Justification for classification or non-classification

An increase of slightly to moderately malaligned sternebrae at the high dose level (300 mg/kg bw/day) was reported in the offspring, which does not seem to be linked to the observed maternal toxicity: the reduced body weight at Day 21 postcoitum, and reduced body weight gain and food consumption.

Since the only adverse effect obeserved in the fetuses were slight to moderate skeletal malformations in the high dose group, diacetone acrylamide is classified in Reproductive toxicity cat 2; H361 Suspected of damaging the unborn child.