4-hydroxybutyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

4-hydroxybutyl acrylate was negative in the Ames Assay

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vitro, other

Remarks:

in vitro cytogenicity / chromosome aberration study & micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please see for more information the read-across justification in Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

42 % survival at 15 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: severe toxicity > 45 µg/mL, with S9: severe toxicity > 160 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cell survival: 53% at 14 ug/mL, 22% at 16 ug/mL and 17% at 18 ug/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: cell survival: ca. 34 % at 16 ug/mL, 23 % at 22ug/mL, 16 % at 24 ug/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at least one dose that inhibited cell growth by 50%

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Species / strain:

other: Syrian hamster embryo fibroblasts

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Result read-across CAS No. 818-61-1

Remarks:

Chromosome Aberration Test

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please see for more information the read-across justification in Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 15 µg/mL 42-45 % survival

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

30 µg/mL without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

in both monolayer and suspension assay

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

15 - 20.5 ug/mL (suspension assay), 55 - 80 ug/mL (monolayer assay)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cell surival: ca 50 % at 14 ug/mL, ca. 20 % at 22 ug/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

12.5 - 25 ug/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Result read-across CAS No. 818-61-1

Remarks:

Mouse lymphoma assay

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his, trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from male Wistar rats livers (induces with phenobarital and ß-naphthoflavone)

Test concentrations with justification for top dose:

1st experiment: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (all strain; SPT with and without S9 mix)
2nd experiment: 0; 10; 33; 100; 333; 1000 and 2500 μg/plate (all strian; PIT with and without S9 mix)
No mutagenicity was observed in the standard plate test. Due to toxicity, the doses were adjusted in the preincubation test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous solvents (ultrapure water)
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, water was used as vehicle.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine

Remarks:

Sterility control: Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
: 3 test plates per dose or per control

Standard plate test: The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al.
Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control; 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control; 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Preincubation Test: The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al.
0.1 mL test solution, vehicle or positive control, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Evaluation criteria:

Acceptance criteria:Generally, the experiment was considered valid if the following criteria were met:
The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
The sterility controls revealed no indication of bacterial contamination.
The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies compatible with the range of the historical positive control data or above.
Fresh bacterial culture containing approximately 109 cells per mL were used.

Assessment criteria: The test substance was considered positive in this assay if the following criteria were met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

TOXICITY: A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ revertants) was observed in the standard plate test depending on the strain and test conditions at and above 1000 μg/plate. In the preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at and above 333 μg/plate.

Decreased revertant numbers were observed at following concentrations (μg/plate):

S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
Without	1000 - 5000	5000	1000 - 5000	2500 - 5000	-
With	2500 - 5000	5000	5000	1000 - 2500	-
Without	2500	-	1000 - 2500	1000 - 2500	1000
with	1000 - 2500	-	1000 - 2500	1000 - 2500	333 - 2500

- = no adverse effects observed

Reduced background growth was observed at following concentrations (μg/plate):

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-SPT	Without	5000	5000	5000	5000	5000
1st-SPT	With	5000	5000	5000	5000	5000
2nd-PIT	Without	2500	2500	2500	2500	-
2nd-PIT	with	-	-	-	-	-

- = no adverse effect observed

Conclusions:

Under the experimental conditions of this study, the test substance 4-Hydroxybutyl Acrylate is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo, HEA did not induce mutagenic effects in rat bone marrow cells in a chronic inhalation study. Furthermore, HEA did neither induce gene mutations nor chromosomal damage in atransgenic rodent mutation assay according to OECD TG 488.

In addition, a micronucleus test with the structural analogue 2-hydroxypropyl acrylate using the oral route in NMRI mice was negative. Based on the present results, it is unlikely that HBA is mutagenic in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vitro mammalian somatic cell study & germ cell study / cytogenicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please see for more information the read-across justification in Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

600 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Result read-across CAS No. 25584-83-2

Remarks:

micronucleus assay

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: Result read-across CAS No. 818-61-1

Remarks:

chromosome aberration assay

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

At 2100 ppm 1/2 mice died after 24 and 48 hr, respectively.

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: Result read-across CAS No. 96-33-3

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The 4 days of exposure caused clinical signs of dyspnoea, bloody discharge from eyes and nose and decrease in body weight (6 - 7 %).

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

other: Result read-across CAS No. 141-32-2 (rat)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The 4 days of exposure caused clinical signs of dyspnoea, disequilibrium, bloody discharge from eyes and nose, mortality of 4/10 animals and decrease in body weight (20 %).

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

other: Result read-across CAS No. 141-32-2 (hamster)

Reference 2

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30.07.2020 to 17.03.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

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. ECHA requested a study according OECD TG 489 (Comet Assay) in rats on the following tissues: liver, glandular stomach and duodenum.
 
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.

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

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)

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

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

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.

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.

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.

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.

 

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Hydroxypropylacrylate
- Analytical purity: 97.8 %
- Batch No. 790201167; Lot No. 32114707

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelman, D-33178 Borchen
- Assigned to test groups randomly: yes
- Housing: 5 animals of identical sex/cage
- Diet (ad libitum): pelleted standard diet (Altromin, D-32791 Lage/Lippe)
- Water (ad libitum): tap water
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 55±10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: CMC (carboxymethyl cellulose)
- Amount of vehicle: 33 mL/kg bw

Duration of treatment / exposure:

single doses

Frequency of treatment:

once

Post exposure period:

not applicable

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

5 animals/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

- Route of administration: single i.p. injection of 10 mL/kg bw cyclophosphamide in 0.9 % NaCl.
- Doses / concentrations: 30 mg/kg b.w

Tissues and cell types examined:

One bone marrow smear was prepared per animal from the tissue cleared from each femur.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
An initial experiment to determine the toxicity of the test substance was conducted. Three male and three female mice were administered the test
substance orally at 1000 mg/kg b.w. This dose resulted in only slight toxicity and was therefore chosen as the top dose. In the main experiment,
two animals died within the first 6 hours of dosing at 1000 mg/kg b.w. so a dose of 600 mg/kg b.w. was chosen as the highest dose that could be used for analysis of micronuclei. All 10 mice at 1000 mg/kg b.w. died within 24 hours of dosing.


TREATMENT AND SAMPLING TIMES:
Five males and five females from each group were sacrificed 24 hours after dosing. Forty eight hours after dosing five animals per sex from the 600 mg/kg dose level were killed.

DETAILS OF SLIDE PREPARATION:
Stained smears were examined by light microscopy for incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. To describe a cytotoxic effect, the ratio of polychromatic to normochromatic erythrocytes was assessed by the examination of at least 1000 erythrocytes.

Evaluation criteria:

Evaluation of Results:
Cells were evaluated for large (aneugenic effects) and small (clastogenic effects) micronuclei. The test substance was classified as mutagenic if it induced either a statistically significant (Mann-Whitney test), dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible, statistically significant positive response for at least one of the test points.

Statistics:

Mann-Whitney test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

600 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

The ratio of normochromatic to polychromatic erythrocytes was slightly affected by the treatment with 2-hydroxypropyl acrylate at a dose of 600 mg/kg bw (at 24 and 48 hours in male mice and at 48 hours in female mice). At this dose level, only slight toxic effects, as evidenced by reduced spontaneous reactivity, were obtained up to 6 hours after dosing. There was no increase in the frequency of micronuclei at any dose level at either 24- or 48-hours after dosing compared to the negative control group.

The positive control compound, cyclophosphamide, produced significantly increased frequencies of micronucleated polychromatic and normochromatic erythrocytes.

 

Following are the results:

 

Males sacrificed at 24 hours:

	Mean Micronuclei/2000 PCE
Dose group	All (%)	Small (%)	Mean PCE/NCE
Negative control	3.2 (0.16)	2.8 (0.14)	1000/873.6
600 mg/kg bw	4.4 (0.22)	3.8 (0.19)	1000/1056.8
300 mg/kg bw	5.4 (0.27)	5.4 (0.27)	1000/1177.6
100 mg/kg bw	4.8 (0.24)	3.8 (0.19)	1000/974.6
Positive control	20.2 (1.01)	18.8 (0.94)	1000/739.6

 

 

Females sacrificed at 24 hours:

 

	Mean Micronuclei/2000 PCE
Dose group	All (%)	Small (%)	Mean PCE/NCE
Negative control	3.2 (0.16)	2.8 (0.14)	1000/737.4
600 mg/kg bw	2.8 (0.14)	2.0 (0.10)	1000/854.6
300 mg/kg bw	5.2 (0.26)	4.8 (0.24)	1000/773.8
100 mg/kg bw	3.2 (0.16)	2.8 (0.14)	1000/918.8
Positive control	19.6 (0.98)	18.4 (0.92)	1000/688.6

 

 

Males and Females sacrificed at 48 hours:

 

	Mean Micronuclei/2000 PCE
Dose group	All (%)	Small (%)	Mean PCE/NCE
600 mg/kg bw males	2.2 (0.11)	2.0 (0.10)	1000/986.2
600 mg/kg bw females	2.2 (0.11)	1.8 (0.09)	1000/1065.4

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro Studies

Bacterial systems

4-Hydroxybutyl acrylate was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. Tested in strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA at concentrations from 33 - 5000 µg/plate (SPT) and 10 - 5000 µg/plate (PIT) with and without metabolic activation. No precipitation of the test substance was observed with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions at and above 333 µg/plate. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. The test substance 4-Hydroxybutyl Acrylate is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF, 2021).

4-Hydroxybutyl acrylate was not mutagenic in Salmonella typhimurium strains TA 98, TA100, TA1535, and TA1537 up to concentrations of 5000 µg/plate (BASF, 1989). The substance was tested in the plate incorporation and preincubation assay with and without metabolic activation (rat liver S-9 mix). Cytotoxicity was observed depending on the strain and test conditions from about 500 µg - 5000 µg/plate.

Mammalian cell gene mutation tests and Cytogenicity tests

No reliable studies concerning genetic toxicity in mammalian cells were identified for 4-hydroxybutyl acrylate (HBA). However, data from the structural analogues 2-hydroxyethyl acrylate (HEA, CAS No. 818-61-1) and hydroxypropyl acrylate (HPA, CAS No. 25584-83-2) are considered appropriate for the assessment.

Data from the structural analogue 2-hydroxyethyl acrylate (HEA, CAS No. 818-61-1):

Dearfield et al. (1989) reported that HEA produced a clear dose-response related increase in mutant frequency in the mouse lymphoma cell assay (L5178Y, TK+/-) (0, 10, 15, 20 and 25 µg/mL) up to 707 mutants per million surviving cells at a concentration of 18 µg/mL (survival 13 %) without metabolic (S9) activation.The majority of the mutant colonies were small colonies indicating the occurrence of chromosomal aberrations. The same authors also reported a dose related increase (0, 15, 18 and 20 µg/mL) in chromosomal aberrations and micronuclei in L5178Y mouse lymphoma cells treated with HEA in the absence of metabolic (S9) activation (Dearfield et al. 1989). The compound demonstrated significant cytotoxicity with a linear dose response. An increase in CA was observed only at cytotoxic concentrations at which the relative total growth was less than 50 % of the control value (e.g. greater than 50 % growth inhibition). It has been demonstrated from several earlier reports that small colony mutant formation in cultured mouse lymphoma cells appears to represent chromosomal alterations to chromosome 11, which carries the tk locus, and that large colonies represent smaller scale, perhaps single gene mutations (Hozier et al. 1981, 1985; Moore et al. 1985). A previous study on the smaller molecular weight monofunctional acrylate/methacrylate esters (Moore et al.1988) demonstrated that this group of compounds induces primarily small-colony mutants, supported by clastogenic activity scored as aberrations in mouse lymphoma cells in vitro. The results with the current set of acrylate/methacrylate esters investigated by Dearfield et al. (1989) are consistent with this induced small-colony/clastogenicity mechanism. This supports the hypothesis that the in vitro genotoxicity of this group of compounds including 2 -hydroxyethyl acrylate acts via a direct acting clastogenic mechanism (as no metabolic activation is required for the positive results) (Dearfield et al. 1989).

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro in a study according to OECD 476 and in compliance with GLP (BASF, 2017). Four experiments were carried out, both with and without metabolic activation (phenobarbital and ß-naphthoflavone induced) at concentrations up to 70 µg/mL. More specifically the following concentrations were tested:

1st Exp: 2.5, 5, 10, 20, 30, 40 and 50 µg/ml (with and without S9 mix) (4hour)

2nd + 3rdExp: 1.87, 3.75, 7.50, 15, 25, 35 and 50 µg/ml (without S9 mix) and 3.75, 7.5, 15, 25, 35, 45 and 60 µg/ml (with S9 mix) (4 hour)

4th Exp: 5, 10, 20, 30, 40, 50, 60 and 70 µg/ml (with S9 mix) (4 hour)

Cytotoxicity was observed in this assay, at concentrations from 40 µg/ml onward (without S9 mix) and from 45 µg/mL onward (with S9 mix). From the data of this study it was concluded that in the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Data from the structural analogue hydroxypropyl acrylate (HPA, CAS No. 25584-83-2):

A mammalian cell (CHO V79) mutation assay conducted with HPA according to OECD TG 476 was negative with and without metabolic activation at concentrations up to 150 µg/mL. Cytotoxicity was observed in this assay, however, at concentrations as low as 30 µg/mL (Evonik Roehm GmbH, 1995). Hydroxypropyl acrylate was tested in a cytogenetic assay with Chinese hamster V79 cells according to OECD TG 473 at concentrations up to 100 µg/mL and was positive with and without metabolic activation in this assay (Evonik Roehm GmbH, 1995). In this assay cytotoxicity was observed only at the highest test concentration. In a chromosomal aberration assay with CHO cells, hydroxypropyl acrylate tested at concentrations up to 240 µg/mL was positive for chromosomal damage. Reduced cell counts were observed at all concentrations (3 to 75 % of control) with relative cell counts for the 15, 30 and 45 µg/mL scored groups of 78, 62, and 45 %, respectively for the nonactivated cultures and 73, 70 and 58 % for the 100, 140 and 160 µg/mL cultures with metabolic activation. An approximate 5 -fold and 10 -fold increase in chromosomal aberrations was observed at 30 and 45 µg/mL, respectively in the non-activated cultures. An approximate 10 -fold increase occurred at 140 µg/mL (but no change was observed at 100 or 160 µg/mL) with metabolic activation, i.e. no dose-dependency was observed (Evonik Roehm GmbH, 2000).

Other (meth)acrylates, including acrylic acid, methyl (meth)acrylate, ethyl (meth)acrylate, 2- ethylhexyl acrylate, and several multifunctional (meth)acrylates (Moore and Doerr, 1990), have been evaluated in in vitro mutagenicity assays with mammalian cells. In these studies, positive results were reported at concentrations that led to a clearly reduced cell survival rate. Studies have indicated that there is an association between chromosomal aberrations and cytotoxicity at exposure concentrations which reduce cell growth to less than 50 % of the control value (Galloway, 2000 and references cited therein). These data suggest that the increase in mutagenicity reported in the chromosomal aberration assays including the micronucleus assay in vitro with HEA and HPA may be an artifact of the experimental method.

Data from the structural analogue Methyl acrylate (MA, CAS No. 96-33-3):

Methyl acrylate did not induce increases in mutant frequencies in the Chinese Hamster Ovary (CHO) HGPRT test performed in the absence of metabolic activation (Moore 1991), and no mutagenicity was demonstrated in AS52/XPRT Chinese hamster cells in which the hgprt gene has been largely deleted and replaced by a single copy of the functional xanthine-guanine phosphoribosyl transferase (XPRT) gene from E. coli. This test was also performed only in the absence of metabolic activation (Oberly 1993).

In contrast, methyl acrylate was active at clearly cytotoxic concentrations (≤50% cell survival) in the Mouse Lymphoma TK+/- mutation assay using L5178Y cells in the absence of metabolic activation (Moore 1988, 1989). The majority of the mutant colonies were small colonies, suggesting that methyl acrylate did act via a clastogenic mechanism (Moore 1988, 1989, Amtower 1986).

There is a number of chromosome aberration tests in vitro available for MA (Ishidate 1981, Moore et al. 1988, 1989). Assays were performed with CHL cells, L5178Y mouse lymphoma cells and CHO cells with (Ishidate 1981) and without metabolic activation (Ishidate 1981, Moore et al. 1988, 1989). All assays gave positive or equivocal results at doses which reduced cell survival to 50 % or lower. There were no doses tested for chromosome aberrations which resulted in 60 % cell survival or more. Thus, there is no experimental evidence that MA might cause chromosome aberrations at non-cytotoxic doses.

More recent studies have indicated that there is an association between chromosomal aberrations and cytotoxicity at exposure concentrations which reduce cell growth to less than 50% of the control value (Galloway, 2000 and references cited therein). These data suggest that the increase in mutagenicity reported in the cytogenicity assays with methyl acrylate may be an artifact of the experimental method.

Conclusion:

In vitro, methyl acrylate was negative in a variety of studies for point mutation both in the presence (Ames test only) and in the absence of metabolic activation, but induced chromosome aberrations in Chinese hamster cells, Chinese ovary cells and L5178Y mouse lymphoma cells in the absence of metabolic activation.

Data from the structural analogue n-butyl acrylate (n-BA, CAS No. 141-32-2):

In a cytogenetic study with Syrian hamster embryo (SHE) fibroblasts, no induction of micronuclei was observed at concentrations of 0.5 - 10 μg/mL without metabolic activation (Wiegand et al., 1989). At the tested concentrations no cytotoxicity of the test substance was observed.

In an in vitro UDS-Test with Syrian hamster embryo fibroblasts, butyl acrylate was tested in concentrations from 1- 400 μg/mL. No induction of unscheduled DNA synthesis in SHE-cells was observed (Wiegand et al., 1989).

In vivo studies

No reliable studies concerning genetic toxicity in vivo were identified for 4-hydroxybutyl acrylate (HBA). However, data from the structural analogues 2-hydroxyethyl acrylate (HEA, CAS No. 818-61-1) and hydroxypropyl acrylate (HPA, CAS No. 25584-83-2) are considered appropriate for the assessment.

Data from the structural analogue hydroxypropyl acrylate (HPA, CAS No. 25584-83-2)

A mouse micronucleus assay was carried out according to OECD TG 474 and GLP regulations with hydroxypropyl acrylate (HPA) using NMRI mice (5 males and 5 females per group) and administering single gavage doses of 0, 100, 300 and 600 mg/kg body weight, respectively. The ratio of normochromatic to polychromatic erythrocytes was slightly affected by the treatment with hydroxypropyl acrylate at a dose of 600 mg/kg bw (at 24 and 48 hours in male mice and at 48 hours in female mice). At this dose level, only slight toxic effects, as evidenced by reduced spontaneous reactivity, were obtained up to 6 hours after dosing. There was no increase in the frequency of micronuclei in bone marrow cells at any dose level at either 24- or 48-hours after dosing compared to the negative control group (Evonik Roehm GmbH, 2000).

Data from the structural analogue 2-hydroxyethyl acrylate (HEA, CAS No. 818-61-1)

HEA did neither induce gene mutations nor chromosomal damage in a transgenic rodent mutation assay according to OECD TG 488

As part of the chronic inhalation study (exposure to 0.5 and 5 ppm HEA; 6 h/day, 5 days/week) some of the rats (i.e. 4 rats/sex/dose group) were sacrificed after 12-months exposure and the bone marrow cells were examined for chromosomal damage. No evidence of chromosomal damage was seen at either dose level (Rampy et al., 1978; Dow Chemical Co., 1979).

Data from the structural analogue Methyl acrylate (MA, CAS No. 96-33-3):

Methyl acrylate has been tested in three in vivo micronucleus assays. It did not induce micronuclei in bone marrow cells of male ddY mice exposed for 3 hours to atmospheres containing 1300 or 2100 ppm (4.64 or 7.50 mg/L) methyl acrylate. Bone marrow samples were taken at 18, 24, 30, 48 or 72 hours after exposure. (The group size was not specified) (Sofuni 1984).

Data from the structural analogue n-butyl-acrylate (n-BA, CAS No. 141-32-2):

Butyl acrylate was tested in two cytogenetic assays in vivo. In those studies, Sprague-Dawley rats and Chinese hamsters were exposed by inhalation 6 hours per day for 4 consecutive days to vapour concentrations of 820 ppm and 817 ppm (corresponding to approx. 4.3 and 4.28 mg/L), respectively. Clear signs of toxicity (dyspnoea, bloody discharge from eyes and nose, decrease in body weight, lethality) were observed in both species. The chromosome analysis carried out in the bone marrow of the animals after the 4-day inhalation did not indicate any chromosome-damaging effect of butyl acrylate in either species or sex (BASF AG 1978, Engelhardt & Klimisch 1983).

Conclusion

In vitro, 4-hydroxybutyl acrylate was negative in the Ames Assay in tester strains TA 98, TA100, TA1535, and TA1537 with and without metabolic activation. Concerning genetic toxicity in mammalian cells in vitro and in vivo, data from the structural analogues 2-hydroxyethyl acrylate and hydroxypropyl acrylate were considered appropriate for the assessment. HEA ist not a mutagenic substance in the HPRT locus assay using CHO cells (with and without metabolic activation) 2-Hydroxyethyl acrylate induced small colonies in the mouse lymphoma cell assay (L5178Y, TK+/-) indicative of a clastogenic effect as well as chromosome aberrations and micronuclei in L5178Y mouse lymphoma cells in the absence of metabolic activation. Hydroxypropyl acrylate was negative in the HPRT assay in Chinese hamster V79 cells with and without metabolic activation. However, the substance induced chromosomal aberrations in V79 and CHO cells both in the presence and absence of metabolic activation at concentrations leading to cytotoxicity. In vivo, a micronucleus test with hydroxypropyl acrylate using the oral route in NMRI mice was negative. In addition, no evidence of chromosomal damage was found in bone marrow cells from rats of the 12-months interim sacrifice as part of a chronic inhalation study with 2-hydroxyethyl acrylate. Furthermore, HEA did neither induce gene mutations nor chromosomal damage in a transgenic rodent mutation assay according to OECD TG 488

Thus, the test substances can be regarded as not mutagenic in vivo. Due to the structural similarities, the same result can be expected for 4-hydroxybutyl acrylate.

References

Galloway (2000). Environ. Mol. Mutagenesis 35:191-201

Hozier et al. (1981). Mutat. Res. 84: 169-181

Hozier et al. (1985). Mutat. Res. 147: 237-242

Moore et al. (1985). Mutat. Res. 151: 161-174

Moore et al. (1988). Environ. Mol. Mutagenesis 11: 49-63

Moore et al. (1989). Mutagenesis 4: 394-403.

Moore and Doerr (1990). Mutagen. 5:609-614.

Moore et al. (1991). Mutagenesis. 6 (1): 77-85.

Ishidate et al. (1981). Gann Monograph on Cancer Research 27: 95-108.

Oberly et al. (1993). Mutation Research 319: 179-187.

Sofuni et al. (1984). Bull National Institute of Hygiene Sciences 102:84-90.

Wiegand et al. (1989). Arch. Toxicol. 63:  250-251.

BASF AG (1978). XXVI/352

Justification for classification or non-classification

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No 1272/2008. Based on the data, classification for genetic toxicity is not warranted under Regulation (EC) No 1272/2008.