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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2020 - March 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to IUCLID chapter 13.2 "Other assessment reports" for the read-across justification.

In their decision on compliance check ECHA requested a further in vivo genotoxicity study to follow up the concern on gene mutation and chromosomal aberrations.
 
Assessment of the biological relevance of in vitro positive mutation studies is usually achieved by performing an in vivo follow up study. The in vivo follow up studies which can be used are the in vivo micronucleus assay for the determination of clastogenic and aneugenic potential of a compound. For the in vivo assessment of gene mutations and chromosome damage (clastogenicity) two guideline conform assays can be used, namely the in vivo comet assay as well as the in vivo transgenic rodent assay (TGR).
 
The in vivo comet assay is a genotoxicity test detecting and quantifying single and double strand breaks. This assay is not a true mutation assay and is regarded as an indicator test, since the fate of the cell with the DNA damage is not considered. The TGR is a true mutation assay, since the detected mutants represent survivors of a mutagen exposure. The TGR assay using the GPT model with the read outs using the GPT as well as SPI-modules is able to detect both mutations on gene level (GPT module) as well as deletion process representing chromosome breakage (SPI-module). Hence both assays are able to detect DNA alterations on the gene and chromosome level. However, the TGR assay is a true mutation assay and less prone to confounding factors (e.g. cytotoxicity). Thus, the preferred in vivo follow up assay for the detection of gene and chromosome mutations is the TGR assay. 
Therefore, the registrant conducted a gene mutation assay (gpt assay and Spi- assay) with transgenic mice (gpt delta mouse) according to OECD TG 488 to assess the potential of Hydroxyethyl acrylate to induce gene point mutation and deletion mutations using the gpt gene (gpt assay) and the red/gam genes (Spi- assay) in the liver and stomach.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)
Version / remarks:
2013
GLP compliance:
yes
Type of assay:
transgenic rodent mutagenicity assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxyethyl acrylate
EC Number:
212-454-9
EC Name:
2-hydroxyethyl acrylate
Cas Number:
818-61-1
Molecular formula:
C5H8O3
IUPAC Name:
2-hydroxyethyl acrylate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: PAU 0118813

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Refrigerator (KS)

Test animals

Species:
mouse
Strain:
C57BL
Remarks:
6JJmsSlc-Tg (gpt delta)
Details on species / strain selection:
C57BL/6JJmsSlc-Tg (gpt delta) mice are commonly used as transgenic animals, and animals of this strain are readily available in in vivo gene mutation assays.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Japan SLC, Inc.
- Age at study initiation: 9 weeks of age
- Weight at study initiation: ca.25 g
- Assigned to test groups randomly: yes
Animals were assigned to groups based on their body weights on Day 1 using LATOX-F/V5 computer system package. Unassigned animals were excluded from the study on Day 1 and will be treated as surplus animals.
- Housing: Animals were housed individually in a plastic cage (W 18.2 × D 26.0 × H 12.8 cm) with bedding (ALPHA-dri™; Shepherd Specialty Papers).
- Diet: pellet diet CRF-1 (Oriental Yeast) ad libitum
- Water: tap water from water bottles ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26
- Humidity (%): 35 to 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: 0.5 w/v% carboxymethyl cellulose sodium salt aqueous solution (0.5 w/v% CMC)

Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing volume (mL) was set at 0.1 mL per 10 g of body weight. The individual dosing volume (mL) was calculated on the basis of the most recent individual body weight measured.

Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Post exposure period:
none
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
275 mg/kg bw/day
No. of animals per sex per dose:
7
Control animals:
yes, concurrent vehicle
Positive control(s):
Benzo[a]pyrene (B[a]P)
The 37.5 mg of B[a]P was weighed, transferred into a graduated test tube and suspended to 3 mL with olive oil to make a 12.5 mg/mL solution. The positive control solution was prepared just before use. The dose was 125 mg/kg bw/day. The positive control substance was administered to animals orally once daily for 5 consecutive days by using a disposable syringe with a Teflon sonde.

Examinations

Tissues and cell types examined:
Liver: Liver is a major site of xenobiotic metabolism.
Stomach: Stomach is the site of first contact, since administration is oral. The histopathological finding was observed in the stomach in dose range-finding study [BSRC’s Exp. No. I919 (652-023)].
Kidney, heart, bladder, lymph node (mesenteric), epididymis and testis were addtionally examined histopathologically by the Sponsor’s demand.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION
In a dose range-finding study [BSRC’s Exp. No. I919 (652-023)] performed in C57BL/6JJmsSlc mice treated with 0, 50.0, 100, 150, 200, 400 or 600 mg/kg bw/day of test substance in 0.5 w/v% CMC for a period of 14 days, 3/3 mice in the 600 mg/kg bw/day group died. No clinical signs of toxicity were observed in the 400 mg/kg bw/day or lower groups. In the histopathological examination, hyperkeratosis and squamous cell hyperplasia were observed in the forestomach in the 200 and 400 mg/kg bw/day groups, and inflammatory cell infiltration and erosion/ulcer with penetration were observed in the forestomach in the 400 mg/kg bw/day group.

TREATMENT AND SAMPLING TIMES

Administration period and manifestation time
[Negative control group and test substance treated groups]
Administration period: Day 1 to 28
Manifestation time: Day 29 to 31
The organs were removed 3 days after the last dosing (Day 31).
[Positive control group]
Administration period: Day 6 to 10
Manifestation time: Day 11 to 24
The organs were removed 14 days after the last dosing (Day 24).

DETAILS ON STUDY DESIGN
Individual body weights of the animals in the negative control group and test substance-treated groups were measured on Day 1 (day of assignment to groups), 3, 8, 15, 22, 29 and 31 (just before organ removal). In the positive control group, the individual body weights of the animals were measured on Day 1, 3, 8, 15, 22 and 24 (just before organ removal). Dead animal was weighed when the condition was found.
The food weight (containing the feeder) of each animal in the negative control group and test substance-treated groups were measured on Day 1, 3, 8, 15, 22 and 29, and the mean daily food consumptions (g/day) was calculated.
In the administration period, animals were observed for clinical signs twice daily. Then, animals were observed for clinical signs once daily until organ removal.

Removal, macroscopic observation, organ weight and storage of organs (tissues)

The animals were necropsied after euthanasia by exsanguination under isoflurane anesthesia. The liver, stomach, kidney, heart, bladder, lymph node (mesenteric), vas deferens/cauda epididymis and testis were removed from each animal. The organ weights of the liver and stomach were measured in grams (to 2 decimal places). The organ weight to body weight ratio (relative organ weight) was calculated from the body weight weighed on the day of necropsy and organ weight (absolute organ weight/final body weight × 100). The organs were removed and stored according to the following methods. The mouse preventer was set at the entrance of the dissecting-room.

Liver: Approximately 5-mm slice (1 horizontal piece) was cut from the left lateral lobe and fixed in an adequate volume of 10 vol% neutral buffered formalin solution. Two samples were prepared from the remaining parts of left lateral lobe using a biopsy trephine (BP-50F, Kai) and separately put into microtubes and frozen in liquid nitrogen (LN2). The other lobes were put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2.
Stomach: The greater curvature of the stomach was incised. The stomach contents were removed by washing with physiological saline. The stomach piece (included forestomach and glandular stomach) was cut to about 4 × 10 mm size and stuck on a thick paper to avoid the curl of the stomach tissue. This part was fixed in an adequate volume of 10 vol% neutral buffered formalin solution. The remaining part was separated into 3 piece (included forestomach and glandular stomach) and put into storage bag and frozen in LN2.
Kidney: The capsule of left kidney was removed and sliced in a thickness of approximately 1 to 2 mm (approximately 2 horizontal pieces in total). Each slice was separately put into microtube and frozen in LN2. The capsule of right kidney was removed and fixed in an adequate volume of 10 vol% neutral buffered formalin solution. The remaining parts were put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2.
Heart: Heart was put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2.
Bladder: The contents were removed by washing with physiological saline. Bladder was put into microtube and frozen in LN2.
Lymph node (mesenteric): About one-third of mesenteric lymph node was fixed in an adequate volume of 10 vol% neutral buffered formalin solution. All remainder mesenteric lymph nodes were put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2.
Germ cells: The right and left testis were put into a microtube separately and frozen in LN2.
The vas deferens/cauda epididymis was cut a little and be placed in a Petri dish containing 1.5 mL of cold Dulbecco’s phosphate-buffered saline (PBS). The germ cells suspended in this PBS were filtered using a cell strainer (pore size 40 µm). About 1 mL of cell suspension was put into microtube and frozen in LN2.

The frozen tissues were stored in an ultra-low temperature freezer.


Pathological examination
Pathological examinations consisted of macroscopic examination and histopathological examination.

Macroscopic examination (necropsy)
The external surface and orifices were observed, followed by observation of the organs and tissues in the abdominal, thoracic, pelvic and cranial cavities. All macroscopic abnormalities were recorded as to the location, size, color tone, etc.

Fixation
The liver, stomach, kidney lymph node (mesenteric) were fixed in an adequate volume of 10% neutral buffered formalin solution as described.

Preparation of the histopathological specimens
Specimens for histopathological examination were prepared for liver and stomach from all animals in negative control group and all test substance-treated groups (excpet animal No. 1302). Fixed tissue samples were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H.E.) according to the routine method.

Histopathological examination
The histopathological specimens were examined microscopically, and all histopathological findings including types and severity were recorded.

Extraction of genomic DNA (details on buffers and medium presented in "Any other information on materials and methods")
Extraction of genomic DNA in the liver and stomach were conducted.
Three milliliters of the buffer for tissue breakage (containing RNase) were poured into a Dounce-type homogenizer and cooled with ice. Each frozen tissue sample was put into the homogenizer and homogenized with a pestle. The homogenized tissue fragments were poured gently into an ice-cooled 15-mL centrifuge tube containing 3 mL of 0.5 mol/L sucrose solution, and centrifuged (centrifuge LC-122, Tomy Seiko) at 3000 rotations/min (1750 G) for 10 minutes. The supernatant was removed and 3 mL of cooled RNase-containing Dounce buffer were added to the tube and mixed well (nuclear/cell suspension).
Then, 3 mL of proteinase K solution were added to the nuclear/cell suspension and gently mixed by inversion. This suspension was incubated at 50°C for about 2 hours until it became clear. The same volume (about 6 mL) of Ph/Cl mixture was added to the solution and mixed by inversion a few times, mixed by using a rotator for 10 minutes, and finally centrifuged (centrifuge LC-122) at 2500 rotations/min (1220 G) for 10 minutes. Next, the upper layer (water layer) was gently collected and transferred into another 15-mL centrifuge tube by a transfer pipette. This procedure was repeated twice (volume of Ph/Cl mixture was same as the removed water layer). After removal of the water layer, the same volume of chloroform/isoamyl alcohol mixture was poured into the tube. The contents were mixed by inversion a few times, mixed by using a rotator for 10 minutes, and finally centrifuged at 2500 rotations/min for 10 minutes. Then, the water layer was transferred into another 50-mL centrifuge tube. Genomic DNA was extracted by gradually adding ethanol in the tube. Extracted genomic DNA was transferred into a microtube containing 70% ethanol and stood for about 10 minutes. The contents were centrifuged (centrifuge MX-160) at 13000 rotations/min (13230 G) for 10 minutes. After the supernatant was removed as much as possible using a micropipette, the tube was stood at room temperature to evaporate ethanol. An appropriate volume (100 µL) of TE buffer (lot No. 02548F, Nippon Gene) was added to the tube. The tube was stood overnight at room temperature to dissolve DNA residues. The DNA solution was stored in a refrigerator after preparation. The concentration of DNA in the genomic DNA solution was measured using a spectrophotometer (NanoDrop® ND-1000, AGC TECHNO GLASS) and adjusted to about 300 to 600 µg/mL with the TE buffer.

Preparation of test strains (for gpt assay)
Thirty milliliters of LB broth, 300 µL of maltose solution (200 mg/mL) and 30 µL of kanamycin solution (20 mg/mL) were poured into a 200-mL baffled Erlenmeyer flask. A suspension (50 µL) of Escherichia coli strain YG6020 that had been thawed after being frozen at -80°C was inoculated into the flask. And it was incubated overnight (about 15 to 16 hours) at 37°C with a shaker at 120 strokes/min as the pre-incubation culture.
One hundred milliliters of LB broth, 1 mL of maltose solution (200 mg/mL) and 100 µL of kanamycin solution (20 mg/mL) were poured into a 500-mL baffled Erlenmeyer flask. The pre-incubation culture (1.5 mL) was inoculated into the flask and it was incubated for about 2 to 3 hours (OD: about 0.9) under the same conditions as the pre-incubation. Then, the bacterial suspension was centrifuged at 2000 rotations/min for 10 minutes. The supernatant was removed and the cells were suspended in LB broth (the volume half of the bacterial suspension collected) containing 10 mmol/L magnesium sulfate (E. coli suspension).

Preparation of test strains (for Spi- assay)
Thirty milliliters of LB broth was poured into a 200-mL baffled Erlenmeyer flask. Each suspension (50 µL) of Escherichia coli [XL-1 Blue MRA, XL-1 Blue MRA (P2) or WL95 (P2))] that had been thawed after being frozen at -80°C was inoculated into the flask. And they were incubated overnight (15 to 16 hours) at 37°C with a shaker at 120 strokes/min as the pre-incubation culture.
One hundred milliliters of LB broth and 1 mL of maltose solution (200 mg/mL) were poured into a 500-mL baffled Erlenmeyer flask. Each pre-incubation culture (1.5 mL each) was inoculated into the flask and they were incubated for about 2 to 3 hours (OD: about 0.9 to 1.0) under the same conditions as the pre-incubation. Then, the bacterial suspensions were centrifuged at 2000 rotations/min for 10 minutes. The supernatant was removed and the cells were suspended in LB broth (the volume half of the bacterial suspension collected) containing 10 mmol/L magnesium sulfate (E. coli suspension). The E. coli suspensions [XL-1 Blue MRA and XL-1 Blue MRA (P2)] were stored on ice or in a refrigerator and used within 3 days. The other E. coli suspension [WL95 (P2)] was stored on ice or in a refrigerator and used within that day.

Packaging of genomic DNA (common to the gpt and Spi- assays)
Packaging was conducted according to the instruction manual attached to Transpack packaging extract (lot No. 0006517934 or 0006543965, Agilent Technologies).
Three red tubes per animal (in gpt assay) or one red tube per animal (in Spi- assay) of Transpack packaging extract were thawed. Using a pipette, 10 µL of genomic DNA solution was transferred to each red tube. The packaging reaction was mixed by pipetting and the tube was incubated at 30°C for 90 minutes. Next, a blue tube of Transpack packaging extract was thawed, and 10 µL of it was transferred to the red tube containing a packaging reaction, and mixed in the same manner. It was incubated at 30°C for another 90 minutes, and diluted up to 100 µL (total 300 µL in gpt assay) or 300 µL (Spi- assay) of SM buffer, and mixed (packaged DNA sample).

Plating of packaged DNA sample (gpt assay)
E. coli (YG6020 strain) suspension 200 µL was dispensed into each tube [2 tubes for calculating total number of colonies (for titering), 5 tubes for calculating mutant frequency (for selection)]. LB broth containing 10 mmol/L magnesium sulfate 495 µL was dispensed into a tube for dilution. Then, 5 µL of the packaged DNA sample was added to the tube for dilution and mixed (diluted suspension). The diluted suspension 5 µL was added to the 2 tubes for titering and mixed. About 60 µL of the packaged DNA was added to the 5 tubes for selection and mixed. The tubes for titering and selection were incubated at 37°C for approximately 20 minutes without shaking and subsequently at 37°C for 30 minutes with a shaker at 120 strokes/min. Then, 2.5 mL of top agar was added to the tube for titering and mixed. The contents were poured on an M9+Cm agar plate. To the tube for selection, 2.5 mL of 6TG top agar was added and the contents were poured on an M9+Cm+6TG agar plate. The agar plates for titering were incubated in an incubator (ILL-60 or SSV-R11DA, Ikeda scientific) at 37°C for 3 days. The agar plates for selection were incubated in the incubator at 37°C for 5 days.
In this single packaging procedure, the total number of colonies per animal reached 300000. Therefore, no further packaging procedure was required.

Plating of packaged DNA sample (Spi- assay)
E. coli (XL-1 Blue MRA) suspension 200 µL was dispensed into each tube [2 tubes for calculating total number of plaques (for titering)]. E. coli [XL-1 Blue MRA (P2)] suspension 200 µL was dispensed into each tube [2 tubes for calculating mutant frequency (for selection)]. LB broth containing 10 mmol/L magnesium sulfate 495 µL was dispensed into a tube for dilution. Then, 5 µL of the packaged DNA sample was added to the tube for dilution and mixed (diluted suspension). The diluted suspension 5 µL was added to the 2 tubes for titering and mixed. About 150 µL of the packaged DNA sample was added to the 2 tubes for selection and mixed. The tubes for titering and selection were incubated at 37°C for approximately 20 minutes without shaking. Then, 2.5 mL of λ-trypticase top agar was added to the tube for titering and selection, and mixed. The contents were poured on a λ-trypticase agar plate. The agar plates were incubated overnight an incubator (SSV-R11DA, Ikeda scientific) at 37°C.
The above packaging procedure was repeated until the total number of plaques per animal reached 300000.

Colony counting (gpt assay)
The number of colonies was counted manually after the incubation (5th days after the incubation). However, gpt mutant candidate colonies on the agar plate for the selection were marked during incubation (on the 3rd and 4th days), because the colonies were not easily distinguished from the precipitation of 6TG on 5th days after the incubation.

Plaque counting (Spi- assay)
The number of plaques was counted manually after the incubation.

Confirmation of mutant phenotypes (gpt assay)
All gpt mutant candidate colonies on the agar plate for the selection were picked up by sterile toothpicks. After the tip of toothpick was rinsed well with 50 µL of 1/15 mol/L Na-K buffer, suspended cells were streaked on the M9+Cm agar plate first and on the M9+Cm+6TG agar plate next. The streaked parts on the agar plates were identified by attaching a cross-section paper and writing a serial number. The plates were incubated in an incubator (ILL-60, Ikeda scientific) at 37°C for 2 days. Only gpt mutant candidate colonies growing on both agar plates were called mutant colonies.

Confirmation of Spi- phenotypes (Spi- assay)
E. coli [XL-1 Blue MRA, XL-1 Blue MRA (P2) and WL95 (P2)] suspension 200 µL was dispensed into each tube. Then, 2.5 mL of λ-trypticase top agar was added to the tube and mixed. The contents were poured on a λ-trypticase agar plate. The agar plates were stood for about 1 hour at room temperature to vaporize the surface.
All Spi- candidates on the agar plate for the selection were punched out with sterilized Pasteur pipette or bore-wide tip. The agar plug was suspended with 50 µL of SM buffer (confirmation solution). One to two microliters of the confirmation solution was spotted on the λ-trypticase agar plates where each of XL-1Blue MRA, XL-1Blue MRA (P2) and WL95 (P2) strains had been spread with λ-trypticase top agar. The spotted parts on the agar plates were identified by attaching a cross-section paper and writing a serial number. The plates were incubated overnight (about 17 to 18 hours) in an incubator (ILL-60, Ikeda scientific) at 37°C. Only Spi- candidates made plaque on the all three agar plates of XL-1Blue MRA, XL-1Blue MRA (P2) and WL95 (P2) strains were called a mutant plaque.

Calculation of total number of colonies (gpt assay)
The number of colonies (N) in the plates for tittering was counted, and then the total number of colonies was calculated using the equations presented in "Any other information on materials and methods".
Evaluation criteria:
-The test substance-treated groups exhibits a statistically significant increase of the mutant frequency compared with the negative control.
-The mutant frequency (mean of group value) in the test substance-treated group is outside the 95% control limit of the laboratory historical negative control data.

If all of the above criteria are met, the test result was considered to be positive. In addition, the biological relevance of the results was taken into consideration for the final judgment.

VALIDITY OF STUDY
Since the following conditions were satisfied, the test was considered successfully performed:
-The mutant frequency in the positive control group markedly increased with a statistically significant difference from the negative control group.
-The mutant frequency in the negative control group was within the acceptable range (95% confidence interval) calculated from the historical data at BSRC.
Statistics:
The data on the mutant frequency from the negative control group and each test substance-treated group were tested by Bartlett’s test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity was determined (not significant on Bartlett’s test), then Dunnett’s multiple comparison test was performed to assess the statistical significance of differences between the negative control group and each test substance-treated group (two-sided, familywise significance level of 0.05). If there was no homogeneity (significant on Bartlett’s test), Steel’s test (two-sided, significance level of 0.05) was performed to analyze the differences.
The data on the mutant frequency from the negative control group and the positive control group were tested by F test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity of variance was determined (not significant on F test), Student’s t test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and the positive control group. If there was no homogeneity (significant on F test), Aspin-Welch’s t test (two-sided, significance level of 0.05) was performed to analyze the differences.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
In the forestomach, hyperkeratosis was observed in all 6 mice in the 275 mg/kg bw/day group.
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mutant frequency (gpt assay)

Liver
In the negative control group, the group mean±SD of mutant frequency among the individuals was 2.46±1.00 (×10-6). The individual values ranged between 1.43-4.05 (×10-6).
The group mean±SD of mutant frequencies in the test substance-treated groups, 25.0, 100 and 275 mg/kg bw/day, were 2.12±0.69 (×10-6), 2.45±0.78 (×10-6) and 2.00±1.39 (×10-6), respectively, and no statistically significant increase was observed compared with the negative control group. The individual values ranged between 0.00-3.55 (×10-6).
All mean values of the negative control and treatment groups were within the 95% control limit range of the historical negative control group data [0.6-4.2 (×10-6)]. The obtained values from the individual animals were also within the 95% control limit of the historical individual control data [0.0-5.6 (×10-6)].
When compared to the negative control group, a statistically significant increase of the positive control group mutant frequency was observed [13.96±3.08 (×10-6)].

Stomach
In the negative control group, the group mean±SD of mutant frequency among the individuals was 3.61±1.53 (×10-6). The individual values ranged between 1.26-5.77 (×10-6).
The group mean±SD of mutant frequencies in the test substance-treated groups, 25.0, 100 and 275 mg/kg bw/day, were 2.86±1.94 (×10-6), 3.06±1.03 (×10-6) and 2.84±1.80 (×10-6), respectively, and no statistically significant increase was observed compared with the negative control group. The individual values ranged between 0.94-6.32 (×10-6).
All mean values of the negative control and treatment groups were within the 95% control limit range of the historical negative control group data [0.0-4.4 (×10-6)]. The obtained values from the individual animals were also within the 95% control limit of the historical individual control data [0.0-5.3 (×10-6)], excpet animal No. 1001 (negative control animal), animal No. 1101 (dose group 25.0 mg/kg bw/day) and animal No. 1305 (dose group 275 mg/kg bw/day).
When compared to the negative control group, a statistically significant increase of the positive control group mutant frequency was observed [21.68±5.61 (×10-6)].

Mutant frequency (Spi- assay)

Liver
In the negative control group, the group mean±SD of mutant frequency among the individuals was 1.84±1.12 (×10-6). The individual values ranged between 0.00-3.28 (×10-6).
The group mean±SD of mutant frequencies in the test substance-treated groups, 25.0, 100 and 275 mg/kg bw/day, were 2.31±2.32 (×10-6), 1.76±1.07 (×10-6) and 2.22±1.60 (×10-6), respectively, and no statistically significant increase was observed compared with the negative control group. The individual values ranged between 0.00-5.65 (×10-6).
All mean values of the negative control and treatment groups were within the 95% control limit range of the historical negative control group data [0.0-5.7 (×10-6)]. The obtained values from the individual animals were also within the 95% control limit of the historical individual control data [0.0-7.6 (×10-6)].
When compared to the negative control group, a statistically significant increase of the positive control group mutant frequency was observed [13.99±2.59 (×10-6)].

Stomach
In the negative control group, the group mean±SD of mutant frequency among the individuals was 2.81±1.93 (×10-6). The individual values ranged between 0.00-5.42 (×10-6).
The group mean±SD of mutant frequencies in the test substance-treated groups, 25.0, 100 and 275 mg/kg bw/day, were 2.84±1.84 (×10-6), 2.11±1.31 (×10-6) and 2.38±1.53 (×10-6), respectively, and no statistically significant increase was observed compared with the negative control group. The individual values ranged between 0.00-5.70 (×10-6).
All mean values of the negative control and treatment groups were within the 95% control limit range of the historical negative control group data [0.0-4.4 (×10-6)]. The obtained values from the individual animals were also within the 95% control limit of the historical individual control data [0.0-5.7 (×10-6)].
When compared to the negative control group, a statistically significant increase of the positive control group mutant frequency was observed [14.16±4.62 (×10-6)].

Body weight and general conditions
There were no statistically significant differences in animal body weights between the negative control group and each of the test substance-treated groups during the administration period.
In the 275 mg/kg bw/day group, one animal (animal No. 1302) was dead on Day 5. In other animals, no clinical signs of toxicity was observed in any of the test substance-treated groups.

Food consumption
There were no statistically significant differences in food consumption between the negative control group and each of the test substance-treated groups.

Organ weight and relative organ weight
The absolute stomach weight and relative stomach weight were statistically significantly increased in the 275 mg/kg bw/day group when compared to the negative control group.
There were no statistically significant differences in the absolute liver weight and relative liver weight between the negative control group and each of the test substance-treated groups.

Gross findings (necropsy)
There were no macroscopic findings related to test substance-treatment in any of the test substance-treated groups.
Black in the spleen was observed in one animal in the 100 mg/kg bw/day group and one animal in the 275 mg/kg bw/day group. This finding was considered to be a spontaneous lesion because it was also found in the negative control group.

Histopathological findings
In the forestomach, hyperkeratosis was observed in all 6 mice in the 275 mg/kg bw/day group.
There were no findings related to test substance treatment in the liver and glandular stomach.

Applicant's summary and conclusion

Conclusions:
Considering all information available, including statistical analysis, it was concluded that Hydroxyethyl acrylate did not induce gene mutation in the liver or stomach of transgenic mice (negative) under the conditions in this study.
Executive summary:

A gene mutation assay (gpt assay and Spi- assay) with transgenic mice (gpt delta mouse) was conducted to assess the potential of Hydroxyethyl acrylate to induce gene point mutation and deletion mutations using the gpt gene (gpt assay) and the red/gam genes (Spi- assay) in the liver and stomach.

The test substance was administered to male transgenic mice orally, once a day, for 28 consecutive days by gavage at the dosage levels of 25.0, 100, and 275 mg/kg bw/day.  After 3 days of manifestation period, the mutant frequencies in the liver and stomach were determined.  One of seven mice in the 275 mg/kg bw/day group died on Day 5. Furthermore, the animals in the 275 mg/kg bw/day group showed a reduced bowy weight gain which corresponded to approx. 80% of that of the vehicle control, This observation was, however, not statistically significant. In the histopathological examination, hyperkeratosis was observed in the forestomach in the 275 mg/kg bw/day group. The average forestomach weight in this group also showed a statistically significant increase as compared to the vehicle control group.

The negative control group values obtained for all organs were within the acceptable range of the historical control data and thus considered as valid.  In the gpt assay and Spi- assay, the mutant frequencies in the liver and stomach of the animals treated with Hydroxyethyl acrylate did not show any increases as compared to the concurrent negative control value.  All group values were also within the historical control data.  In some cases the individual values surpassed the upper limit of the 95% control limit of the historical data.  However, these increases are considered as not biologically relevant, since their distribution was sporadic and the increase was also observed in the control group. 

The mutant frequencies in the liver and stomach in the positive control group, which was treated with benzo[a]pyrene, were increased and these increases were statistically significant compared with those of the negative control group.  Therefore, the present study was judged to be properly conducted.