Reactionmass of octan-2-yl prop-2-enoate and octan-3-yl prop-2-enoate and octan-4-yl prop-2-enoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

MTDID 44428 was tested in an Ames assay and the results of two in vitro genetic toxicity studies were read-across from the structural analog IOA (source chemical) to MTDID 44428 (target chemical). The results of the studies were:

Ames Assay: Negative with and without metabolic activation when tested according to OECD 471.

Chromosome aberration study: Clastogenic solely at cytotoxic concentrations without metabolic activation at 48 hour exposure when tested according to OECD 473.

Mouse Lymphoma assay: Negative with and without metabolic activation when tested according to OECD 476.

Based on the results of the studies, the criteria for classification are not met.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Remarks:

No deviations ocurred that negatively impacted the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 3M Company, Lot 11
- Purity, including information on contaminants, isomers, etc.: 99.7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Kept in a room with controls set to maintain 19°C to 25°C

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding):
The test article was suspended in 200 proof ethanol.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
The test article was suspended in 200 proof ethanol.

Target gene:

Tryptophan and histidine operons

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:

The Aroclor 1254-induced rat liver S9 fraction (Lot No. 4048) was purchased from Molecular Toxicology, Inc. (Boone, NC). The metabolic activation mixture was prepared fresh on each day of testing and maintained on ice throughout the assay.
The S9 mixture (7.5% [v/v]) for metabolic activation consists of the following in a total volume of 10 mL:
0.75 mL of S9 fraction
0.20 mL of MgCl2 (0.4M) - KCl (1.65M)
0.05 mL of glucose-6-phosphate (1M)
0.40 mL of nicotinamide adenine dinucleotide phosphate (NADP) (0.1M)
5.00 mL of phosphate buffered saline
3.60 mL of sterile distilled deionized wate

Test concentrations with justification for top dose:

In the range-finding assay, MTDID 44428 was tested in single plates at 1, 5, 10, 50, 100, 500, 1000, and 5000 µg/plate with strains TA100 and WP2 uvrA using the plate incorporation method. Precipitates were not observed at any concentration in both strains with and without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥ 500 µg/plate in TA100 without metabolic activation and ≥ 1000 µg/plate in TA100 with metabolic activation.

In the mutagenicity assay, MTDID 44428 was tested 5, 10, 25, 50, 100, 250, 500, and 1000 µg/plate in strains TA1537, TA98, TA1535, and TA100, and 100, 250, 500, 1000, 2500, and 5000 µg/plate in strain WP2 uvrA, using the plate incorporation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 200 proof ethanol

- Justification for choice of solvent/vehicle: Test article solubility and test system compatibility.

- Justification for percentage of solvent in the final culture medium: Per OECD 471 recommendation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene, ICR-191 Acridine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10^8 and 10^9 cells per mL of culture
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Stock cell solutions were incubated overnight prior to dosing to bring the cell density to the appropriate level needed for testing.
- Exposure duration/duration of treatment: 48 hours with the test article in agar.
- Harvest time after the end of treatment (sampling/recovery times): Plates were counted following a 48 hour exposure period.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 hours
- Selection time (if incubation with a selective agent): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours
- Selective agent: Histidine and trypotophan-minimal agars.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 10^8 and 10^9 cells per mL of culture

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
The test substance was considered positive for mutagenicity if it induced an increase of revertants per plate with increasing concentration. The increases should be at least 2 times the vehicle control background frequency for strains with high spontaneous levels (i.e., TA100) and 3 times for those with low spontaneous levels (TA1537, TA98, TA1535, and WP2 uvrA). These increases should be seen in at least 2 or more successive concentrations or the response should be repeatable at a single concentration.

Rationale for test conditions:

Per OECD 471.

Evaluation criteria:

Criteria for a Positive Response:

The test substance was considered positive for mutagenicity if it induced an increase of revertants per plate with increasing concentration. The increases should be at least 2 times the vehicle control background frequency for strains with high spontaneous levels (i.e., TA100) and 3 times for those with low spontaneous levels (TA1537, TA98, TA1535, and WP2 uvrA). These increases should be seen in at least 2 or more successive concentrations or the response should be repeatable at a single concentration.

Criteria for a Negative Response:

The test substance was considered to be negative for inducing mutagenicity if it did not induce a response which fulfills the criteria for a positive response.

Statistics:

For each concentration level and for each condition, the mean revertant count and standard deviation (SD) were determined.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity at 250 ug/plate and greater without metabolic activation and 1000 ug/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity at 100 ug/plate and greater without metabolic activation and 500 ug/plate and greater with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity at 100 ug/plate and greater without metabolic activation and at 500 ug/plate and greater with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity at 500 ug/plate and greater without metabolic activation and 1000 ug/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 4.4
- Data on osmolality: No data
- Possibility of evaporation from medium: Not expected based on analytical conducted on dosing solutions.
- Water solubility: the average measured water solubility of the 2-octyl acrylate isomer is 4.98 mg/L (RSD=21%), the average water solubility of the 3-octyl acrylate isomer is 5.24 mg/L (RSD=19%), and the average water solubility of the 4-octyl acrylate isomer is 4.35 mg/L (RSD=16%) at 24.2°C
- Precipitation and time of the determination: No precipitation was observed.

RANGE-FINDING/SCREENING STUDIES (if applicable): See "Test concentrations with justification for top dose" field.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Vehicle and positive controls performed as expected and were within historical ranges for the laboratory.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : No increase in revertant colonies was observed.
- Statistical analysis: For each concentration level and for each condition, the mean revertant count and standard deviation (SD) were determined.

Ames test:
- Signs of toxicity : Cytotoxicity was observed at ≥ 100 µg/plate in strains TA1537 and TA100 without metabolic activation; at ≥ 250 µg/plate in strain TA1335 without metabolic activation; and at ≥ 500 µg/plate in strains TA98 without metabolic activation and TA1537 and TA100 with metabolic activation. Cytotoxicity was also observed at 1000 µg/plate in strains TA98 and TA1535 with metabolic activation. No cytotoxicity was observed with strain WP2 uvrA with or without metabolic activation.
- Individual plate counts : See attached table.
- Mean number of revertant colonies per plate and standard deviation : See attached table.

Conclusions:

MTDID 44428 is negative in the bacterial reverse mutation assay (Ames assay) in the presence and absence of metabolic activation (S9).

Executive summary:

MTDID 44428 was evaluated for mutagenic activity in the in vitro bacterial reverse mutation assay. Four tester strains of Salmonella typhimurium(TA1537, TA98, TA100, and TA1535) and Escherichia coli strain (WP2uvrA) were used for mutagenicity testing. The study was conducted according to OECD 471 in compliance with OECD GLP. MTDID 44428 was prepared as a stock formulation in 200 proof ethanol at concentrations up to 50 mg/mL for each assay. Mutagenicity testing was performed in triplicate at each concentration with and without an Aroclor 1254-induced rat liver S9 metabolic activation system. In the range-finding assay, MTDID 44428 was tested in single plates at 1, 5, 10, 50, 100, 500, 1000, and 5000 µg/plate with strains TA100 and WP2uvrA using the plate incorporation method. Precipitates were not observed at any concentration in both strains with and without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥ 500 µg/plate in TA100 without metabolic activation and ≥ 1000 µg/plate in TA100 with metabolic activation. In the mutagenicity assay, MTDID 44428 was tested 5, 10, 25, 50, 100, 250, 500, and 1000 µg/plate in strains TA1537, TA98, TA1535, and TA100, and 100, 250, 500, 1000, 2500, and 5000 µg/plate in strain WP2 uvrA, using the plate incorporation method. Precipitates were not observed at any concentration, in any strain with or without metabolic activation. Cytotoxicity was observed at ≥ 100 µg/plate in strains TA1537 and TA100 without metabolic activation; at ≥ 250 µg/plate in strain TA1535 without metabolic activation; and at ≥ 500 µg/plate in strains TA98 without metabolic activation and TA1537 and TA100 with metabolic activation. Cytotoxicity was also observed at 1000 µg/plate in strains TA98 and TA1535 with metabolic activation. No cytotoxicity was observed with strain WP2 uvrA with or without metabolic activation. In the mutagenicity assay, criteria for a negative response were met for all tester strains with and without metabolic activation. The data from the vehicle and positive controls demonstrated the validity and sensitivity of this test system for detecting chemical mutagens with and without metabolic activation. These data support the conclusion that MTDID 44428 is negative for mutagenic activity in the S. typhimurium strains TA1537, TA98, TA100, and TA1535 and in the E. coli strain WP2 uvrA, with and without metabolic activation, under the conditions of this assay. 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target chemical (MTDID 44428, CASRN 87015-11-0) and the source chemical (isooctyl acrylate (IOA) CAS 29590-42-9) are isomers that contain the same functional acrylate moiety attached to primarily C8 alkyl hydrocarbon chains (C7-C9, C8 rich for the source chemical) with variable branching. The source chemical differs from the target chemical in both the degree and position of branching of the alkyl ester group with the source chemical being branched at the terminal end of the alkyl chain while the target chemical is branched near the acrylate functional group. The acrylate group is expected to be metabolized in the same manner in both substances and the remaining alkyl chain will be metabolized and excreted via the same pathway. The source chemical and target chemical have the same molecular weight and very similar log Kow values (Target: 4.7-4.8, Source: 4.5-4.7). Similar ADME profiles are expected between the two substances as the metabolic pathway of acrylate esters has been well characterized. Acrylate and methacrylate functionalities are electrophilic and both may participate in Michael addition reactions. Metabolism is expected to occur through the same pathways, hydrolysis by carboxylesterases into two metabolites, an alcohol and an acrylic acid moiety with minor conjugation to gluthathione. Hydrolysis is similar across the acrylate family and enhances the elimination of the chemical upon exposure (McCarthy & Witz, 1997). Studies with n-butyl acrylate and 2-ethylhexyl acrylate confirm that the acrylic acid metabolite enters aerobic oxidation and in completely metabolized to CO2 with only a minor proportion be conjugated to glutathione and excreted in the urine as a N-acetyl cysteine conjugate (Sanders, JM et. al, 1988; Gut, I, et al. 1988). The previously mentioned studies have also demonstrated that enzymatic hydrolysis kinetic constants for methacrylate and acrylate esters are similar.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Target Chemical
The target molecule, MTDID 44428 (CASRN 87015-11-0), is a multi-constituent substance defined as the reaction mass of octan-2-yl acrylate, octan-3-yl acrylate and octan-4-yl acrylate as represented by the following structures.
(see attached read-across justification document).
Molecular weight of the target chemical is 184.3
Source Chemical:
The source chemical, isooctyl acrylate (IOA) CAS 29590-42-9, is defined as a UVCB and is represented by the following structure (see attached read-across justification document).
The average molecular weight of the source substance is ca. 184.0. The source chemical differs from the target chemical in the degree and position of branching of the alkyl ester group with the source chemical having variable methyl branching along the alkyl chain while the target chemical is branched immediately adjacent to the acrylate functional group.

Purity and Impurities :
MTDID 44428 is a multi-constituent substance and the three acrylate constituents contribute >99% of the content. There are very low levels of residual reactants and reaction side products.
Isooctyl acrylate is a UVCB substance, based on the mixed-isomer nature of the material. As a UVCB substance, all components are considered part of the substance and the concept of impurities has little meaning. Acrylate ester content of IOA is >99 %, with very low levels of residual reactants and reaction side products. These non-acrylate components are substantially similar and do not impact the read-across of test results from IOA.
3. ANALOGUE APPROACH JUSTIFICATION
Analogue Approach Justification

The target chemical and source chemical are closely related alkyl acrylate compounds. They differ slightly in the structure of the alkyl ester portion of the molecule. MTDID 44428 contains a methyl, ethyl or propyl branch at the 1-carbon of the alkyl chain portion of the molecule, which is always C8 in total. IOA may have methyl or ethyl branches at one or more positions along the alkyl ester group. The carbon chain is predominantly C8 in total with lesser contributions of C7 and C9 (C8 on average).
The environmental toxicity of low molecular weight acrylate esters (aquatic mortality and immobilization) is by protein adduct formation via a Michael-type addition mechanism. In the environment, toxicity increases on a molar concentration basis with molecular weight (and concomitantly, hydrophobicity) due to increased ability of the molecule to reach its active site. The excess toxicity is mitigated in high molecular weight acrylate esters with log P > 5.

The mammalian toxicity of IOA and MTDID 44428 is also based on protein adduct formation via a Michael-type addition with the acrylate groups. Both the target and source chemical are weak dermal sensitizers (section 5) indicating that an equivalent mechanism of toxicity is at work for both substances based on identical functional groups and molecular weights and very similar log Kow values and water solubilities.

The number of hydrophobic carbons of IOA relative to MTDID 44428 is predicted to be similar resulting in very similar octanol water partition coefficient values. This was confirmed experimentally and the log Kow for IOA is 4.5-4.7 while the log Kow for MTDID 44428 is 4.7-4.8. Additionally, IOA and MTDID 44428 have very similar water solubility at 12.44 and 14.6 mg/L, respectively. IOA is expected to be metabolized via the same hydrolytic and enzymatic pathways as MTDID 44428, forming acrylic acid and isooctanol.

Similar mammalian metabolic pathways are expected for IOA and MTDID 44428 based on Sanders, et. al and Gut, et al. The source chemical and target chemical have the same molecular weight and very similar log Kow values. Similar ADME profiles are expected between the two substances as the metabolic pathway of acrylate esters has been well characterized. Acrylate and methacrylate functionalities are electrophilic and both may participate in Michael addition reactions. Metabolism is expected to occur through the same pathways, hydrolysis by carboxylesterases into two metabolites, an alcohol and an acrylic acid moiety with minor conjugation to gluthathione. Hydrolysis is similar across the acrylate family and enhances the elimination of the chemical upon exposure (McCarthy & Witz, 1997). Studies with n-butyl acrylate and 2-ethylhexyl acrylate confirm that the acrylic acid metabolite enters aerobic oxidation and in completely metabolized to CO2 with only a minor proportion be conjugated to glutathione and excreted in the urine as a N-acetyl cysteine conjugate (Sanders, JM et. al, 1988; Gut, I, et al. 1988). The previously mentioned studies have also demonstrated that enzymatic hydrolysis kinetic constants for methacrylate and acrylate esters are similar.

As can be seen in the table in section 5, the source and target substances have very similar environmental and mammalian hazard profiles for endpoints where each substance has experimental data. This further supports the hypothesis that the target and source substances are expected to behave similarly in mammalian and environmental systems with the same mechanism of action and that read-across of the data for higher-tier endpoints is appropriate in an effort to reduce unnecessary animal testing.

4. DATA MATRIX
See 'Other Information Including Tables' or attached justification.

Supporting References
Gut, I, Vodička, Cikrt, M, Sapota, A, and Kavan, I (1988) Distribution and elimination of (14C)-2-ehtylheyxyl acrylate radioactivity in rats. Archives of Toxicology 62:346-350.
McCarty, TJ and Witz, G (1997) Structure-activity relationships in the hydrolysis of acrylate and methacrylate esters by carboxylesterase in vitro. Toxicology 116: 153-158.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mammalian cell line, other: See 'Remarks'

Remarks:

Human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

In the absence of S9-mix at the 48-hour exposure time, IOA induced a statistically significant increase in the number of cells with chromosome aberrations at the highest, cytotoxic concentration, both when gaps were included and excluded.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the Chromosome aberration study, IOA did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence of S9-mix at the 3 hour and 24 hour exposure times and in the presence of S9-mix, in the first and second cytogenetic assays. However, in the second cytogenetic assay in the absence of S9-mix at the 48-hour exposure time, IOA induced a statistically significant increase in the number of cells with chromosome aberrations at the highest, cytotoxic concentration, both when gaps were included and excluded. Because this increase was above the historical control data range, observed in both duplicate cultures it was considered biologically relevant and IOA was considered clastogenic.

No effects of IOA on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be concluded that IOA does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in the report.

READ-ACROSS DATA MATRIX

	Target substance	Source substance
CHEMICAL NAME	Reaction mass of octan-2-yl acrylate, octan-3-yl acrylate and octan-4-yl acrylate	Isooctyl acrylate
CAS#	44914-03-6	29590-42-9
Molecular formula	C11H20O2	C11H20O2 (on average)
Molecular Weight	184.3	184.3 (on average)
Melting Point	Experimental: <-35 °C	Experimental: < -90 °C at 1004 hPa
Boiling Point	Experimental: 217.6 °C (normalized)	Experimental: 196.8 °C at 1016 hPa
Density	Experimental: 0.8665 at 23 °C	Experimental: 0.885 g/cm3 at 20.0 °C
Vapour Pressure	Experimental: .06 hPa at 18 °C	Experimental: 1 hPa at 20 °C
Partition Coefficient (log KOW)	Experimental: 4.7-4.8	Experimental: 4.5 - 4.7
Water Solubility	Experimental: Individual isomers had solubilites of 4-5 mg/L, total was 14.6 mg/L	Experimental: 12.44 mg/L at 23.1 °C
Stability in Water	Experimental: t1/2at 25 °C, pH 9, 37.7-116 days t1/2at 25 °C, pH 7, 137 days - not determinable t1/2at 25 °C, pH 4, 154 days - not determinable. Hydrolysis product could be detected at pH 9 but not pH 7 and 4. Half-life increased from 2-octyl < 3-octyl < 4-octyl isomers.	Adaptation, readily biodegradable
Aerobic Biodegradation	Experimental: 54.7% after 28 days, biodegradation essentially stopped at day 11 (OECD 301F)   67% after 28 days. No residual material could be detected in test chambers on day 28. In abiotic control, residual test material was 4.8% of initial result (OECD 302C)	Experimental: 93-95% after 28 days (OECD 301D)
Bioconcentration		Not bioaccumulative (Extensive metabolism)
Transport and Distribution	Experimental: Koc 630 (OECD121)	Experimental: Koc 650-3900 (OECD121)
Henry's Law constant	NDA	Experimental: 1780 Pa*m3/mol at 23.1 °C
Acute Toxicity to Fish  (P. promelasunless noted)	NDA	Experimental: 96-hour LC50 0.67 mg/L (OECD 202)
Chronic Toxicity to Fish	NDA	Waived
Acute Toxicity to Aquatic Invertebrates (D. magna)	NDA	Experimental: 48-hour EC50 0.4 mg/L (OECD 202)
Long-Term Toxicity to Aquatic Invertebrates (D. magna)	NDA	Experimental: 28-day NOEC 0.065 mg/L (OECD 202 rev 1984)
Toxicity to Algae and Aquatic Plants (P. subcapitata)	NDA	QSAR result not read across
Toxicity to Microorganisms (activated sludge respiration)	Experimental: 3-hour EC50 >1000 mg/L (OECD 209)	Experimental: 3-hour EC50 >1000 mg/L (OECD 209)
Acute Oral Toxicity	Experimental: Rat oral LD50 > 2,000 mg/kg	Experimental: Rat oral LD50 > 5,000 mg/kg
Acute Dermal Toxicity	Read-across from source: Rabbit dermal LD50 > 2,000 mg/kg	Experimental: Rabbit dermal LD50 > 2,000 mg/kg
Acute Inhalation Toxicity	Read-across from source: NDA	Experimental: NDA
Skin Irritation	Experimental: Irritating (GHS Cat. 2)	Experimental: Not irritating
Eye Irritation	Experimental: Not Irritating	Experimental: Not irritating
Skin Sensitization	Experimental: Weak sensitizer (GHS Category 1B)	Experimental: Weak sensitizer (GHS Category 1B)
Ames Assay	Experimental: Non-mutagenic	Experimental: Non-mutagenic
in vitroChromosome Aberration	Read-across from source: Clastogenic at cytotoxic concentrations	Experimental: Clastogenic at cytotoxic concentrations
in vitroMouse Lymphoma Assay	Read-across from source: Non-mutagenic	Experimental: Non-mutagenic
28 Day Oral Toxicity	Read-across from source: NOAEL = 1,000 mg/kg/day	Experimental: NOAEL = 1,000 mg/kg/day
90 Day Oral Toxicity	Read-across from source: NOAEL = 600 mg/kg/day	Experimental: NOAEL = 600 mg/kg/day
Reproductive/Developmental Screening Study (Dermal)	Read-across from source: NOAEL = 20% Dermal Exposure	Experimental: NOAEL = 20% Dermal Exposure
Prenatal Developmental Study (Oral)	Read-across from source: NOAEL = 1,000 mg/kg/day	Experimental: NOAEL = 1,000 mg/kg/day
Carcinogenicity (Dermal)	Read-Across from source: Not Carcinogenic	Experimental: Not Carcinogenic (5% Dermal Exposure)

Conclusions:

In the Chromosome aberration study, IOA did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence of S9-mix at the 3 hour and 24 hour exposure times and in the presence of S9-mix, in the first and second cytogenetic assays. However, in the second cytogenetic assay in the absence of S9-mix at the 48-hour exposure time, IOA induced a statistically significant increase in the number of cells with chromosome aberrations at the highest, cytotoxic concentration, both when gaps were included and excluded. Because this increase was above the historical control data range, observed in both duplicate cultures it was considered biologically relevant and IOA was considered clastogenic.
 
No effects of IOA on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be concluded that IOA does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in the report.

The source chemical (IOA) is clastogenic in human lymphocytes and the results are read-across to the target substance (MTDID 44428). The clastogenic activitiy is confined to a cytotoxic concentrations and prolonged incubation in the absence of S9-mix.

Executive summary:

The similarities between the structural, physical & chemical, toxicity, and predicted metabolic properties of the source and target substances presented above support the read-across hypothesis for genetic toxicity. The data are adequate and reliable scientific information to support the hypothesis. Therefore, based upon the data and considerations presented in the above sections, it can be concluded that the results of the genetic toxicity study with source substance will accurately predict the results for the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, of the REACH Regulation for the target substance.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July -November 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Study by OECD Guidelines by experienced laboratory

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

During incubation, the temperature dipped briefly to 30.3 C. This effect had no effect on the results of the study.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/ streptomycin (50 U/ml and 50 ìg/ml respectively) and heparin (30 U/ml).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes/no
- Periodically checked for karyotype stability: yes/no
- Periodically 'cleansed' against high spontaneous background: yes/no

Metabolic activation:

with and without

Metabolic activation system:

S9 (9000 x G supernatant)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle:

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Solvent for positive controls: Hanks’ Balanced Salt Solution (HBSS) without calcium and magnesium.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
DURATION
- Exposure duration: 3 hours, 24 hours, 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells):
24 hours/48 hours

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic Index

Species / strain:

mammalian cell line, other: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: at 330 ug/ml

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
other: see conclusion

MTDID-7819 is clastogenic in human lymphocytes. The clastogenic activitiy is confined to a cytoxic concentrations and prolonged incubation in the absence of S9-mix.

Executive summary:

In the assay described above, MTDID-7819 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence of S9-mix at the 3 hour and 24 hour exposure times and in the presence of S(-mix, in the first and second cytogenetic assays. However, in the second cytogenetic assay in the absence of S9-mix at the 48-hour exposure time, MTDID-7819 induced a statistically significant increase in the number of cells with chromosome aberrations at the highest, cytotoxic concentration, both when gaps were included and excluded. Because this increase was above the historical control data range, observed in both duplicate cultures it was considered biologically relevant and MTDID-7819 was considered clastogenic. No effects of MTDID-7819 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be concluded that MTDID-7819 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in the report.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target chemical (MTDID 44428, CASRN 87015-11-0) and the source chemical (isooctyl acrylate (IOA) CAS 29590-42-9) are isomers that contain the same functional acrylate moiety attached to primarily C8 alkyl hydrocarbon chains (C7-C9, C8 rich for the source chemical) with variable branching. The source chemical differs from the target chemical in both the degree and position of branching of the alkyl ester group with the source chemical being branched at the terminal end of the alkyl chain while the target chemical is branched near the acrylate functional group. The acrylate group is expected to be metabolized in the same manner in both substances and the remaining alkyl chain will be metabolized and excreted via the same pathway. The source chemical and target chemical have the same molecular weight and very similar log Kow values (Target: 4.7-4.8, Source: 4.5-4.7). Similar ADME profiles are expected between the two substances as the metabolic pathway of acrylate esters has been well characterized. Acrylate and methacrylate functionalities are electrophilic and both may participate in Michael addition reactions. Metabolism is expected to occur through the same pathways, hydrolysis by carboxylesterases into two metabolites, an alcohol and an acrylic acid moiety with minor conjugation to gluthathione. Hydrolysis is similar across the acrylate family and enhances the elimination of the chemical upon exposure (McCarthy & Witz, 1997). Studies with n-butyl acrylate and 2-ethylhexyl acrylate confirm that the acrylic acid metabolite enters aerobic oxidation and in completely metabolized to CO2 with only a minor proportion be conjugated to glutathione and excreted in the urine as a N-acetyl cysteine conjugate (Sanders, JM et. al, 1988; Gut, I, et al. 1988). The previously mentioned studies have also demonstrated that enzymatic hydrolysis kinetic constants for methacrylate and acrylate esters are similar.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Target Chemical
The target molecule, MTDID 44428 (CASRN 87015-11-0), is a multi-constituent substance defined as the reaction mass of octan-2-yl acrylate, octan-3-yl acrylate and octan-4-yl acrylate as represented by the following structures.
(see attached read-across justification document).
Molecular weight of the target chemical is 184.3
Source Chemical:
The source chemical, isooctyl acrylate (IOA) CAS 29590-42-9, is defined as a UVCB and is represented by the following structure (see attached read-across justification document).
The average molecular weight of the source substance is ca. 184.0. The source chemical differs from the target chemical in the degree and position of branching of the alkyl ester group with the source chemical having variable methyl branching along the alkyl chain while the target chemical is branched immediately adjacent to the acrylate functional group.

Purity and Impurities :
MTDID 44428 is a multi-constituent substance and the three acrylate constituents contribute >99% of the content. There are very low levels of residual reactants and reaction side products.
Isooctyl acrylate is a UVCB substance, based on the mixed-isomer nature of the material. As a UVCB substance, all components are considered part of the substance and the concept of impurities has little meaning. Acrylate ester content of IOA is >99 %, with very low levels of residual reactants and reaction side products. These non-acrylate components are substantially similar and do not impact the read-across of test results from IOA.
3. ANALOGUE APPROACH JUSTIFICATION
Analogue Approach Justification

The target chemical and source chemical are closely related alkyl acrylate compounds. They differ slightly in the structure of the alkyl ester portion of the molecule. MTDID 44428 contains a methyl, ethyl or propyl branch at the 1-carbon of the alkyl chain portion of the molecule, which is always C8 in total. IOA may have methyl or ethyl branches at one or more positions along the alkyl ester group. The carbon chain is predominantly C8 in total with lesser contributions of C7 and C9 (C8 on average).
The environmental toxicity of low molecular weight acrylate esters (aquatic mortality and immobilization) is by protein adduct formation via a Michael-type addition mechanism. In the environment, toxicity increases on a molar concentration basis with molecular weight (and concomitantly, hydrophobicity) due to increased ability of the molecule to reach its active site. The excess toxicity is mitigated in high molecular weight acrylate esters with log P > 5.

The mammalian toxicity of IOA and MTDID 44428 is also based on protein adduct formation via a Michael-type addition with the acrylate groups. Both the target and source chemical are weak dermal sensitizers (section 5) indicating that an equivalent mechanism of toxicity is at work for both substances based on identical functional groups and molecular weights and very similar log Kow values and water solubilities.

The number of hydrophobic carbons of IOA relative to MTDID 44428 is predicted to be similar resulting in very similar octanol water partition coefficient values. This was confirmed experimentally and the log Kow for IOA is 4.5-4.7 while the log Kow for MTDID 44428 is 4.7-4.8. Additionally, IOA and MTDID 44428 have very similar water solubility at 12.44 and 14.6 mg/L, respectively. IOA is expected to be metabolized via the same hydrolytic and enzymatic pathways as MTDID 44428, forming acrylic acid and isooctanol.

Similar mammalian metabolic pathways are expected for IOA and MTDID 44428 based on Sanders, et. al and Gut, et al. The source chemical and target chemical have the same molecular weight and very similar log Kow values. Similar ADME profiles are expected between the two substances as the metabolic pathway of acrylate esters has been well characterized. Acrylate and methacrylate functionalities are electrophilic and both may participate in Michael addition reactions. Metabolism is expected to occur through the same pathways, hydrolysis by carboxylesterases into two metabolites, an alcohol and an acrylic acid moiety with minor conjugation to gluthathione. Hydrolysis is similar across the acrylate family and enhances the elimination of the chemical upon exposure (McCarthy & Witz, 1997). Studies with n-butyl acrylate and 2-ethylhexyl acrylate confirm that the acrylic acid metabolite enters aerobic oxidation and in completely metabolized to CO2 with only a minor proportion be conjugated to glutathione and excreted in the urine as a N-acetyl cysteine conjugate (Sanders, JM et. al, 1988; Gut, I, et al. 1988). The previously mentioned studies have also demonstrated that enzymatic hydrolysis kinetic constants for methacrylate and acrylate esters are similar.

As can be seen in the table in section 5, the source and target substances have very similar environmental and mammalian hazard profiles for endpoints where each substance has experimental data. This further supports the hypothesis that the target and source substances are expected to behave similarly in mammalian and environmental systems with the same mechanism of action and that read-across of the data for higher-tier endpoints is appropriate in an effort to reduce unnecessary animal testing.

4. DATA MATRIX
See 'Other Information Including Tables' or attached justification.

Supporting References
Gut, I, Vodička, Cikrt, M, Sapota, A, and Kavan, I (1988) Distribution and elimination of (14C)-2-ehtylheyxyl acrylate radioactivity in rats. Archives of Toxicology 62:346-350.
McCarty, TJ and Witz, G (1997) Structure-activity relationships in the hydrolysis of acrylate and methacrylate esters by carboxylesterase in vitro. Toxicology 116: 153-158.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.1 uL/mL produced complete toxicity in both activated (2%) growth and non-activated cultures (0% growth). Therefore, 0.11 uL/mL was used as the high dose for non-activated cultures and 0.2 uL/mL was used as the high dose for activated cultures.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

READ-ACROSS DATA MATRIX

	Target substance	Source substance
CHEMICAL NAME	Reaction mass of octan-2-yl acrylate, octan-3-yl acrylate and octan-4-yl acrylate	Isooctyl acrylate
CAS#	44914-03-6	29590-42-9
Molecular formula	C11H20O2	C11H20O2 (on average)
Molecular Weight	184.3	184.3 (on average)
Melting Point	Experimental: <-35 °C	Experimental: < -90 °C at 1004 hPa
Boiling Point	Experimental: 217.6 °C (normalized)	Experimental: 196.8 °C at 1016 hPa
Density	Experimental: 0.8665 at 23 °C	Experimental: 0.885 g/cm3 at 20.0 °C
Vapour Pressure	Experimental: .06 hPa at 18 °C	Experimental: 1 hPa at 20 °C
Partition Coefficient (log KOW)	Experimental: 4.7-4.8	Experimental: 4.5 - 4.7
Water Solubility	Experimental: Individual isomers had solubilites of 4-5 mg/L, total was 14.6 mg/L	Experimental: 12.44 mg/L at 23.1 °C
Stability in Water	Experimental: t1/2at 25 °C, pH 9, 37.7-116 days t1/2at 25 °C, pH 7, 137 days - not determinable t1/2at 25 °C, pH 4, 154 days - not determinable. Hydrolysis product could be detected at pH 9 but not pH 7 and 4. Half-life increased from 2-octyl < 3-octyl < 4-octyl isomers.	Adaptation, readily biodegradable
Aerobic Biodegradation	Experimental: 54.7% after 28 days, biodegradation essentially stopped at day 11 (OECD 301F)   67% after 28 days. No residual material could be detected in test chambers on day 28. In abiotic control, residual test material was 4.8% of initial result (OECD 302C)	Experimental: 93-95% after 28 days (OECD 301D)
Bioconcentration		Not bioaccumulative (Extensive metabolism)
Transport and Distribution	Experimental: Koc 630 (OECD121)	Experimental: Koc 650-3900 (OECD121)
Henry's Law constant	NDA	Experimental: 1780 Pa*m3/mol at 23.1 °C
Acute Toxicity to Fish  (P. promelasunless noted)	NDA	Experimental: 96-hour LC50 0.67 mg/L (OECD 202)
Chronic Toxicity to Fish	NDA	Waived
Acute Toxicity to Aquatic Invertebrates (D. magna)	NDA	Experimental: 48-hour EC50 0.4 mg/L (OECD 202)
Long-Term Toxicity to Aquatic Invertebrates (D. magna)	NDA	Experimental: 28-day NOEC 0.065 mg/L (OECD 202 rev 1984)
Toxicity to Algae and Aquatic Plants (P. subcapitata)	NDA	QSAR result not read across
Toxicity to Microorganisms (activated sludge respiration)	Experimental: 3-hour EC50 >1000 mg/L (OECD 209)	Experimental: 3-hour EC50 >1000 mg/L (OECD 209)
Acute Oral Toxicity	Experimental: Rat oral LD50 > 2,000 mg/kg	Experimental: Rat oral LD50 > 5,000 mg/kg
Acute Dermal Toxicity	Read-across from source: Rabbit dermal LD50 > 2,000 mg/kg	Experimental: Rabbit dermal LD50 > 2,000 mg/kg
Acute Inhalation Toxicity	Read-across from source: NDA	Experimental: NDA
Skin Irritation	Experimental: Irritating (GHS Cat. 2)	Experimental: Not irritating
Eye Irritation	Experimental: Not Irritating	Experimental: Not irritating
Skin Sensitization	Experimental: Weak sensitizer (GHS Category 1B)	Experimental: Weak sensitizer (GHS Category 1B)
Ames Assay	Experimental: Non-mutagenic	Experimental: Non-mutagenic
in vitroChromosome Aberration	Read-across from source: Clastogenic at cytotoxic concentrations	Experimental: Clastogenic at cytotoxic concentrations
in vitroMouse Lymphoma Assay	Read-across from source: Non-mutagenic	Experimental: Non-mutagenic
28 Day Oral Toxicity	Read-across from source: NOAEL = 1,000 mg/kg/day	Experimental: NOAEL = 1,000 mg/kg/day
90 Day Oral Toxicity	Read-across from source: NOAEL = 600 mg/kg/day	Experimental: NOAEL = 600 mg/kg/day
Reproductive/Developmental Screening Study (Dermal)	Read-across from source: NOAEL = 20% Dermal Exposure	Experimental: NOAEL = 20% Dermal Exposure
Prenatal Developmental Study (Oral)	Read-across from source: NOAEL = 1,000 mg/kg/day	Experimental: NOAEL = 1,000 mg/kg/day
Carcinogenicity (Dermal)	Read-Across from source: Not Carcinogenic	Experimental: Not Carcinogenic (5% Dermal Exposure)

Conclusions:

Reading-across the results from the source chemical (IOA), the target chemical (MTDID 44428) is not mutagenic in a L5178Y TK +/- Mouse Lymphoma Cell Assay in the presence and absence of metabolic activation.

Executive summary:

The similarities between the structural, physical & chemical, toxicity, and predicted metabolic properties of the source and target substances presented above support the read-across hypothesis for genetic toxicity. The data are adequate and reliable scientific information to support the hypothesis. Therefore, based upon the data and considerations presented in the above sections, it can be concluded that the results of the genetic toxicity study with source substance will accurately predict the results for the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, of the REACH Regulation for the target substance.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1980 - July 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: There may be slight protocol modifications from the current OECD 476 test protocol

Qualifier:

according to guideline

Guideline:

other: Clive, D and Spector JFS. Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y Mouse Lymphoma cells. Mutation Research 31:17-29, 1975.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The test method is believed to be similar to OECD 476 - In vitro cell gene mutation test

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinas (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S-9

Test concentrations with justification for top dose:

Without activation: 0.020, 0.015 0.011, 0.084,0.0063, 0.0048, 0.0036, 0.0027, 0.0020 and 0.0015 ul/ml
With activation: 0.11, 0.084, 0.063, 0.047, 0.036, 0.027, 0.020, 0.015, 0.011 and 0.0084 ul/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: With activation: 7,12-Dimethylbenz(a)anthracene. Without activation: Ethylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 24 and 48 hours
- Selection time (if incubation with a selection agent): 10-12 days
SELECTION AGENT (mutation assays): Trifluorothymidine
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cell density)

Evaluation criteria:

Comparison of mutation frequency of treated plates with vehicle control plates to determine the presence of a significant increase in mutagenicity.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.1 ul/ml produced complete toxicity in both activated (2% growth) and non-activated cultures (0% growth). Therefore, 0.11 ul/ml was used as the high dose for non-activated cultures and 0.2 ul/ml was used as the high dose for activated cultures.

Vehicle controls validity:

valid

Positive controls validity:

valid

The initial cytotoxicity tests demonstrated that approximately 0.1 ul/ml produced complete toxicity in both activated (2% growth) and non-activated cultures (0% growth). Therefore, 0.11 ul/ml was used as the high dose for non-activated cultures and 0.2 ul/ml was used as the high dose for activated cultures.

For the non-activated cultures, all of the clonable cultures , except the culture which received the lowest dose (0.0015 ul/ml) exhibited mutant frequencies which were not significantly higher than those of solvent controls. The 0.0015 ul/ml dose culture exhibited a mutant frequency which was twice that of the average mutant frequencies of the corresponding solvent controls, but since there was no dose response, and the average mutant frequency was the same as the solvent control for the positive control, the mutant frequency was considered to be within the range of experimental error.

For the activated cultures, three of the clonable cultures exhibited mutant frequencies which were twice that of the average mutant frequency of the corresponding solvent controls. However, there was no dose response and the highest mutant frequency did not exceed that of solvent controls for the positive control. Therefore the data points were considered to be within the range of experimental error.

Conclusions:

Interpretation of results (migrated information):
other: Negative with and without metabolic activation. (Criteria used for interpretation : Expert Judgement).

The test article was negative in the L5178Y TK+/- Mouse Lymphoma Mutagenesis assay.

Executive summary:

The test article was tested in the presence and absence of exogenous metabolic activation by Aroclor induced rat liver S-9 in the L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay. Although some of the test cultures exhibited mutant frequencies which were two fold greater than the average mutant frequency of the corresponding solvent control, no dose response was seen and the mutant frequencies were within the range of experimental error (mutant frequencies similar to those for solvent controls for the positive controls). The test article was therefore considered negative in the assay under the test conditions

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)