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

Endpoint summary

Description of key information

The repeated dose toxicity studies from the source chemical (IOA) were read-across to the target chemical (MTDID 44428).

Two Repeat Dose studies, a 28-Day Oral and a 90-Day Oral, have been conducted on IOA.

 

28-Day Oral NOAEL: 1000 mg/kg

 

90-Day Oral NOAEL: 600 mg/kg

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

1. Temporary deviations from the maximum humidity occurred in teh animal room. Lab data do not indicate an effect of the dviations. 2. In the second wek of treatment, test substance used for formulations was inadverrently stored at room temperature in

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Housing:Animals were group housed in macrolon cages with setrilized houseing as bedding and paper as cage enrichment.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to the start of treatment
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-22.0 C
- Humidity (%): 41-78%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light 12 hours light/ 12 hours dark with temporary fluctuations (1 hour max) dues to pupullary rflex tesets and/or ophthalmoscopic exam.):
IN-LIFE DATES: From: 21 August 2009 To: 18 September 2009

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test article solubility in vehicle
- Concentration in vehicle: 10-333 mg/ml resulting in doses of 0 (control), 30,300 and 1000

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing samples were prepared within 6 hours of dosing. Samplese were analyzed on a single occasion during the in-life phase for homgeneity and accuracy of prparation (all concentrations). Accuracy was acceptable is the mean measured concentrations were 90-110% of the target concentration.

Duration of treatment / exposure:

28 Days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:

Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Bsed on results of a 5 day range finding study. Animals in main study were doses at 0 (control), 30, 300 and 1000 mg/kg/day
- Rationale for animal assignment (if not random): random

Positive control:

corn oil

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice Daily
- Cage side observations were included. Yes
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once Daily
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:
OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Collected from all surving animals prior to post mortem exam
- Anaesthetic used for blood collection: Yes iso-flurane
- Animals fasted: Yes
- How many animals: All surviving
- Parameters checked in table [No.1] were examined. Yes
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Same as haematology
- Animals fasted: Yes
- How many animals: all surviving
- Parameters checked in table [No.2] were examined.
URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: grip strength: other: hearing ability, pupillary reflex, static righting reflex,
OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes see Table 3 All tissue samples in groups 1 and 4 (control and hight dose) were examined by the pathologist. In addition, the thyroid, kidneys, stomach and liver in groups 2 and 3 males and females (stomach and liver only for females) were also examined by the pathologist.
HISTOPATHOLOGY: Yes see Table 4

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
BODY WEIGHT AND WEIGHT GAIN
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
FOOD EFFICIENCY
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
OPHTHALMOSCOPIC EXAMINATION
HAEMATOLOGY
CLINICAL CHEMISTRY:
Higher bilirubin in males at 1000 mg/kg
Higher cholesterol levels in females at 300 1000 mg/kg
Lower cholesterol in males at 1000 mg/kg
Higher postssium levels in males and females at 1000 mg/kg
Higher inforganix phosphate in males at 1000 mg/kg
URINALYSIS
NEUROBEHAVIOUR
ORGAN WEIGHTS
Higher liver weight and liver to body weight ration in males at 300(not statistically significant for liver weight) and males and females at 1000 mg/kg
Higher thyroid and thyroid to body weight ratio in males and females at 1000 mg/kg
Higher kidney weight and kidney to body weight ratio in one male (no 17) at 1000 mg/kg
GROSS PATHOLOGY
Kidneys in one male at 300 mg/kg and two males at 1000 mg/kg had pale discoloration of the kidneys. One male at 1000 mg/kg (no 17) had grey-white/dark red, part firm/part soft nodules on teh left kidney which was enlarged in size.
HISTOPATHOLOGY: NON-NEOPLASTIC
HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Stomach: Hyperplasia of teh squamous epithelium of the forestomach was recorded in 2/5 females at 300 mg/kg(minimal) and in 2/5 males (minimal) and 3/5 females (minmal-slight) at 1000 mg/kg.
Liver: Hypertrophy of the hepatoxytes of teh centrilobular are(minimal-slight) was recorded in 4/5 males at 1000 mg/kg
Kidneys: Cortical hyaline droplets in teh kidneys were recorded in 4/5 males at 300 mg/kg (minimal-slight) and 5/5 males at 1000 mg/kg (slight-moderate). This was the microscopic correlate to teh pale kiscoloration recroded at necropsy. The occurrence of cortical hyaline droplets was accompanied by an increased incidence and severity of corticomedullary tubular basophilia in males at 1000 mg/kg (4/5 animals minimal-slight)
Thyroid: A slightly increased incidence and severity of hypertrophy /hyperplasia of teh follicular epithelium as recorded in males at 1000 mg/kg (4/5 animals, moderate-slight)
HISTORICAL CONTROL DATA (if applicable)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Conclusions:

NOAEL of 30 mg/kg established in the study but the effects are rodent specific and of no relevance to human risk assessment. Based on the results of the study, the human-relevant No Observed Adverse Effect Level is 1000 mg/kg-day.

Executive summary:

The repeated-dose oral toxicity potential of MTDID 7819 (clear colorless liquid, lot 4/2009) was evaluated in male and female Wistar rats. METHODS: This study was performed in compliance with OECD GLP (1997). The study design was based on the following guidelines: EC No. 440/2008 B.7 (2008), OECD 407 (2008) and OPPTS 870.3050 EPA 712-C-00-366 (2000). MTDID 7819 was prepared in corn oil (vehicle) within 6 hours prior to dosing. Rats (5/sex/group) received 0 (vehicle), 30, 300 or 1000 mg/kg-day MTDID 7819 via oral gavage for 28 days. Parameters evaluated: clinical observations (daily), functional observations (week 4), body weight (weekly), food consumption (weekly), clinical pathology (termination), macroscopic examination (termination), organ weights and microscopic examination of specific tissues. RESULTS: No toxicologically-relevant changes were noted for clinical observations, functional observations, body weights, food consumption or hematology. Adaptive liver changes included increased liver weights (300 mg/kg-day males and 1000 mg/kg-day males and females); centrilobular hepatocellular hypertrophy (1000 mg/kg-day males); total bilirubin, cholesterol, potassium, and/or inorganic phosphate concentrations (1000 mg/kg-day males and females); and cholesterol changes in 300 mg/kg-day females. Although these changes were adaptive, the report authors considered the magnitude of the liver weight increase in the 1000 mg/kg-day-treated animals (i.e. >20%) to be adverse. Adaptive thyroid changes included increased thyroid weights (1000 mg/kg-day males and females); and increased incidence and severity of thyroid follicular epithelium hypertrophy/hyperplasia (1000 mg/kg-day males). Hyperplasia was noted in the forestomach of 300 mg/kg-day-treated females and males and females at 1000 mg/kg-day. The hyperplasia was considered an adaptive response to local irritation in the forestomach and is a common adaptive phenomenon in rat studies. Hyaline droplet formation was noted in the kidneys of the 300 and 1000 mg/kg-day-treated male rats. This result is attributed to the male rat-specific alpha-2u-globulin protein and is not considered relevant for female rats or higher species, including humans. Nephroblastoma correlated with nodules and higher kidney weight in one 1000 mg/kg-day-treated male rat which was considered spontaneous and not related to the test article. CONCLUSION: Based on the results of this study, the authors proposed a rat NOAEL of 30 mg/kg-day. The findings at 300 mg/kg-day were considered rat-specific and of no relevance to humans. Based on the results of the study, the human-relevant No Observed Adverse Effect Level is 1000 mg/kg-day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

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

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

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 of the study with the source chemical, the target substance (MTDID 44428) has a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day.

Executive summary:

The similarities between the structural, physical & chemical, toxicity, and predicted metabolic propertiesof the source and target substances presented above support the read-across hypothesis for repeated dose oral 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 repeated dose oral 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:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July 2012 to 17 October 2012 (in-life phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC No 440/2008, B.26 Repeated Dose (90 days) Toxicity (oral), 2008. OPPTS 870.3100, EPA 712-C-98-199, 90-Day Oral Toxicity in Rodents, 1998. JCSCL 1973, Notification of Mar. 31 2012 by MHLW (0331 No.7), METI (No. 5) and MOE (No. 110331009)

Deviations:

yes

Remarks:

Slides stained for alpha globulin were incubated for 5 minutes instead of 12. Negative control slides taken from animal no. 31 rather than no. 46. The study integrity was not adversely affected by the deviations.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han) (outbred, SPF-Quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Males: 163-167 g, Females: 128-133 g
- Fasting period before study: None
- Housing: Group housed 5 animals per sex in Macrolon cages (MIV type, height 18 cm)
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF Speziadiaten GmbH, Germany) ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: At least 5 days prior to treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 13 July 2012 To: 17 October 2012

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulation performed at WIL Research Europe and on information from the sponsor. The same vehicle was used in the 28-day oral toxicity study.
- Concentration in vehicle: Dose levels were 40, 150, and 600 mg test article/kg body weight in 3 mL vehicle/kg body weight.
- Amount of vehicle (if gavage): Dose volume was 3 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations, in weeks 1, 6 and 13). Stability in vehicle over 8 days in the refrigerator was also determined (highest and lowest concentration, in week 1).

Duration of treatment / exposure:

Daily dosing by oral gavage for 90 days.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:

Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a previous 28-day oral toxicity study by oral gavage.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Satellite groups not utilized.
- Post-exposure recovery period in satellite groups: Satellite groups not utilized.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily after dosing
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily after dosing.
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study.
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to dosing and at week 13.
- Dose groups that were examined: All animals prior to dosing and Groups 1 and 4 at week 13.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: All
- Parameters checked in Appendix 1 were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of necropsy.
- Animals fasted: Yes, overnight
- How many animals: All
- Parameters checked in Appendix 1 were examined.
URINALYSIS: Yes
- Time schedule for collection of urine: At end of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Appendix 1 were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12
- Dose groups that were examined: Groups 1 and 4
- Battery of functions tested: sensory activity, grip strength, motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Details in Other Information)
HISTOPATHOLOGY: Yes (Details in Other Information)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: No abnormal clinical signs or mortality were observed during the test.
BODY WEIGHT AND WEIGHT GAIN: Body weights and body weight gain of treated animals remained in the same range as controls over the study period.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
FOOD EFFICIENCY: No toxicologically significant changes in food consumption before or after correction for body weight were noted.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
OPHTHALMOSCOPIC EXAMINATION: No toxicologically significant opthamology findings were noted.
HAEMATOLOGY: No toxicologically relevant changes occurred in haematological parameters of treated rats.
CLINICAL CHEMISTRY: No toxicologically relevant changes occurred in clinical chemistry parameters of treated rats.
URINALYSIS: Lower pH in males and females was noted in the 600 mg/kg group. Higher concentration of ketones in females at 600 mg/kg.
NEUROBEHAVIOUR: Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals of the control and 600 mg/kg group.
ORGAN WEIGHTS: Higher liver weight and liver to body weight ratio in males and females at 600 mg/kg (mean liver to body weight ratio 18% higher than control rats). Higher kidney weight in males at 40, 150, and 600 mg/kg and females at 600 mg/kg, and higher kidney to body weight ratio in males and females at 150 and 600 (mean kidney to body weight ratio 10, 16, and 30% higher (males) or 6, 11, and 20% higher (females) than control means at 40, 150, and 600 mg/kg respectively).
GROSS PATHOLOGY: Accentuated lobular pattern of the liver in 4/10 males at 40 mg/kg and 3/10 males and 1/10 females at 600 mg/kg. Enlarged liver in 1/10 males at 150 mg/kg and 4/10 males at 600 mg/kg. Enlarged kidneys in 6/10 males at 600 mg/kg.
HISTOPATHOLOGY: NON-NEOPLASTIC: Kidneys: Cortical hyaline droplets were increased in incidence and severity in all treated groups of males increasing from minimal in 3 control (group 1) males to slight to severe in all ten high dose (group 4) males. Immunohistochemical staining confiremed that these hyaline droplets represented alpha 2 globulin in high dose (group 4) males. Associated with the above findings were granular casts recorded at minimal or moderate degree in two middle dose (group 3) males and at minimal to moderate degree in 6 high dose (group 4) males. Minor degrees (minimal or slight) of corticomedullary tubular basophilia were increased in all treated groups of males. Liver: Diffuse midzonal/centrilobular hypertrophy at a minimal degree was seen in 2 middle dose (group 3) and at minimal or slight degree in 8 high dose (group 4) males.(minimal also in one high dose (group 4) female). Thyroid glands: Diffuse follicular hypertrophy/hyperplasia was recorded at minimal degree in 2 control (group 1), 1 low dose (group 2), 3 middle dose (group 3), and at minimal or slight degree in 5 high dose (group 4) males.
HISTOPATHOLOGY: NEOPLASTIC: None
HISTORICAL CONTROL DATA (if applicable)
OTHER FINDINGS

Dose descriptor:

NOAEL

Remarks:

Male NOAEL=40 mg/kg/day, Female NOAEL=150 mg/kg/day.

Effect level:

ca. 40 - ca. 150 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Male NOAEL=40 mg/kg/day, Female NOAEL=150 mg/kg/day.

Critical effects observed:

not specified

Conclusions:

Based on the presence of granular casts in the kidneys of males at 150 and 600 mg/kg, combined with the higher kidney weights in males and females at 600 mg/kg and in males at 150 mg/kg, a No Observed Adverse Effect Level (NOAEL) for the test article of 40 mg/kg (males) or 150 mg/kg (females) was established. Kidney damage in males was found to have been caused by an increase in alpha 2 globulin which ultimately lead to proximal cortical tubule cell injury, the formation of granular casts, and an increased cell turnover as manifest by an increased incidence/severity of tubular basophilia in males at 40, 150, and 600 mg/kg. This triad of lesions has been referred to as male rat nephropathy syndrome. As alpha 2 globulin is not present in female rats nor in higher mammals, including humans, nephropathy syndrome is considered to be of no risk to humans.

Kidney effects were seen in both studies but histopathology and immunohistochemical staining have shown that these effects are due to male rat nephropathy syndrome and are not relevant to human risk assessment. Because the only adverse effect is caused by the male rat nephropathy syndrome, the NOAEL for purposes of human risk assessment is set at 600 mg/kg.

Executive summary:

The toxicity potential of the test article was evaluated in a 90-day repeat dose study in male and female Crl:WI(Han) rats. The study was conducted under GLP conditions. The test method was based on OECD 408 (1998). The test material was diluted in corn oil (vehicle) to the appropriate dose concentrations. Male and Female rats (10/sex/group) received 0 (vehicle), 40, 150, or 600 mg/kg/day of the test article via oral gavage for 90 days. Clinical observations (daily), body weight (weekly), food consumption (weekly), functional tests (week 12), ophthalmoscopic examination (pretest and week 13), haematology (prior to necropsy), clinical chemistry (prior to necropsy), urinalysis (at end of treatment), gross pathology (at necropsy), histopathology (at necropsy) and immunohistochemistry (at necropsy) were each examined. No abnormal clinical signs or mortality were observed during the study. Functional observations, ophthalmoscopic examination, body weights and body weight gain, food consumption, haematology and clinical chemistry were all normal and no toxicologically relevant changes were noted. Upon urinalysis lower pH in males and females in the 600 mg/kg group was noted. In addition, a higher concentration of ketones was noted in 600 mg/kg group females. Upon gross necropsy, higher liver weight and liver to body weight ratio was noted in males and females at 600 mg/kg (mean liver to body weight ratio 18% higher than control rats). Higher kidney weight was noted in males at 40, 150, and 600 mg/kg and females at 600 mg/kg, and higher kidney to body weight ratio in males and females at 150 and 600 (mean kidney to body weight ratio 10, 16, and 30% higher (males) or 6, 11, and 20% higher (females) than control means at 40, 150, and 600 mg/kg respectively). Accentuated lobular pattern of the liver was noted in 4/10 males at 40 mg/kg and 3/10 males and 1/10 females at 600 mg/kg. Enlarged liver was noted in 1/10 males at 150 mg/kg and 4/10 males at 600 mg/kg. Enlarged kidneys were noted in 6/10 males at 600 mg/kg. Upon histopathological examination of the kidneys, cortical hyaline droplets were increased in incidence and severity in all treated groups of males increasing from minimal in 3 control (group 1) males to slight to severe in all ten high dose (group 4) males. Immunohistochemical staining confirmed that these hyaline droplets represented alpha 2 globulin in high dose (group 4) males. Associated with the above findings were granular casts recorded at minimal or moderate degree in two middle dose (group 3) males and at minimal to moderate degree in 6 high dose (group 4) males. Minor degrees (minimal or slight) of corticomedullary tubular basophilia were increased in all treated groups of males. Upon histopathological examination of the liver, diffuse midzonal/centrilobular hypertrophy at a minimal degree was seen in 2 middle dose (group 3) and at minimal or slight degree in 8 high dose (group 4) males.(minimal also in one high dose (group 4) female). Upon histopatholigical examination of the thyroid glands, diffuse follicular hypertrophy/hyperplasia was recorded at minimal degree in 2 control (group 1), 1 low dose (group 2), 3 middle dose (group 3), and at minimal or slight degree in 5 high dose (group 4) males. Based on the presence of granular casts in the kidneys of males at 150 and 600 mg/kg, combined with the higher kidney weights in males and females at 600 mg/kg and in males at 150 mg/kg, a No Observed Adverse Effect Level (NOAEL) for the test article of 40 mg/kg (males) or 150 mg/kg (females) was established. Kidney damage in males was found to have been caused by an increase in alpha 2 globulin which ultimately lead to proximal cortical tubule cell injury, the formation of granular casts, and an increased cell turnover as manifest by an increased incidence/severity of tubular basophilia in males at 40, 150, and 600 mg/kg. This triad of lesions has been referred to as male rat nephropathy syndrome. As alpha 2 globulin is not present in female rats nor in higher mammals, including humans, nephropathy syndrome is considered to be of no risk to humans.

Kidney effects were seen in both studies but histopathology and immunohistochemical staining have shown that these effects are due to male rat nephropathy syndrome and are not relevant to human risk assessment. Because the only adverse effect is caused by the male rat nephropathy syndrome, the NOAEL for purposes of human risk assessment is set at 600 mg/kg.

Reference 4

Endpoint:

sub-chronic toxicity: oral

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: No abnormal clinical signs or mortality were observed during the test.
BODY WEIGHT AND WEIGHT GAIN: Body weights and body weight gain of treated animals remained in the same range as controls over the study period.
FOOD EFFICIENCY: No toxicologically significant changes in food consumption before or after corr
ection for body weight were noted.
OPHTHALMOSCOPIC EXAMINATION: No toxicologically significant opthamology findings were noted.
HAEMATOLOGY: No toxicologically relevant changes occurred in haematological parameters of treated rats.
CLINICAL CHEMISTRY: No toxicologically relevant changes occurred in clinical chemistry parameters of treated rats.
URINALYSIS: Lower pH in males and females was noted in the 600 mg/kg group. Higher concentration of ketones in females at 600 mg/kg.
NEUROBEHAVIOUR: Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals of the control and 600 mg/kg group.
ORGAN WEIGHTS: Higher liver weight and liver to body weight ratio in males and females at 600 mg/kg (mean liver to body weight ratio 18% higher than control rats). Higher kidney weight in males at 40, 150, and 600 mg/kg and females at 600 mg/kg, and higher kidney to body weight ratio in males and females at 150 and 600 (mean kidney to body weight ratio 10, 16, and 30% higher (males) or 6, 11, and 20% higher (females) than control means at 40, 150, and 600 mg/kg respectively).
GROSS PATHOLOGY: Accentuated lobular pattern of the liver in 4/10 males at 40 mg/kg and 3/10 males and 1/10 females at 600 mg/kg. Enlarged liver in 1/10 males at 150 mg/kg and 4/10 males at 600 mg/kg. Enlarged kidneys in 6/10 males at 600 mg/kg.
HISTOPATHOLOGY: NON-NEOPLASTIC: Kidneys: Cortical hyaline droplets were increased in incidence and severity in all treated groups of males increasing from minimal in 3 control (group 1) males to slight to severe in all ten high dose (group 4) males. Immunohistochemical staining confirmed that these hyaline droplets represented alpha 2 globulin in high dose (group 4) males. Associated with the above findings were granular casts recorded at minimal or moderate degree in two middle dose (group 3) males and at minimal to moderate degree in 6 high dose (group 4) males. Minor degrees (minimal or slight) of corticomedullary tubular basophilia were increased in all treated groups of males. Liver: Diffuse midzonal/centrilobular hypertrophy at a minimal degree was seen in 2 middle dose (group 3) and at minimal or slight degree in 8 high dose (group 4) males.(minimal also in one high dose (group 4) female). Thyroid glands: Diffuse follicular hypertrophy/hyperplasia was recorded at minimal degree in 2 control (group 1), 1 low dose (group 2), 3 middle dose (group 3), and at minimal or slight degree in 5 high dose (group 4) males.
HISTOPATHOLOGY: NEOPLASTIC: None

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See 'Remarks'

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Male Kidney Effects:

Based on the presence of granular casts in the kidneys of males at 150 and 600 mg/kg, combined with the higher kidney weights in males at 600 and 150 mg/kg, a No Observed Adverse Effect Level (NOAEL) for the test article of 40 mg/kg was established. Kidney damage in males was found to have been caused by an increase in alpha 2 globulin which ultimately lead to proximal cortical tubule cell injury, the formation of granular casts, and an increased cell turnover as manifest by an increased incidence/severity of tubular basophilia in males at 40, 150, and 600 mg/kg. This triad of lesions has been referred to as male rat nephropathy syndrome. As alpha 2 globulin is not present in female rats nor in higher mammals, including humans, nephropathy syndrome is considered to be of no risk to humans.

Kidney effects were observed in males but histopathology and immunohistochemical staining have shown that these effects are due to male rat nephropathy syndrome and are not relevant to human risk assessment. Because the only adverse effect is caused by the male rat nephropathy syndrome, the NOAEL for purposes of human risk assessment is set at 600 mg/kg.

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 of the study with the source chemical, the target substance (MTDID 44428) has a No Observed Adverse Effect Level (NOAEL) for purposes of human rusk assessment of 600 mg/kg/day.

Executive summary:

The similarities between the structural, physical & chemical, toxicity, and predicted metabolic propertiesof the source and target substances presented above support the read-across hypothesis for repeated dose oral 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 repeated dose oral 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.

Justification for classification or non-classification

Reading-across the results of the studies conducted on the source substance (IOA), the target substance (MTDID 44428) is not classified as a target organ toxicant after repeated exposure.