Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acrylate

Endpoint summary

Description of key information

IBOA/ target substance

Subacute

NOAEL general systemic toxicity, rat, oral gavage: 100 mg/kg bw/d due to clinical chemistry effects (increased urea; indicative for effects on the hepatobiliar system; IBOA, OECD 422, GLP)

Metabolite and metabolite donor data

subchronic

NOAEL general toxicity, rat, oral, drinking water: 83 mg/kg bw/d due to unspecific effects considered as secondary to unpalatibility of the test item (Acrylic Acid, 90d, pre-guideline)

NOAEL general toxicity, rat, oral, drinking water: 84/111 mg/kg bw/d due to unspecific effects considered as secondary to unpalatibility of the test item (Butyl Acrylate, 90d, similar to OECD 408)

NOAEL (general toxicity, rat, oral, gavage: 15 mg/kg bw/d due to liver effects (Isobornyl Acetate, 13 weeks, similar to OECD 408)

chronic

NOAEL general toxicity, rat, oral, drinking water: 61 mg/kg bw/d due to unspecific effects considered as secondary to unpalatibility of the test item (Acrylic Acid, 12 months, comparable to OECD 452)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-05 - 2012-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identity : Isobornyl acrylate
Alternative name : VISIOMER® IBOA
CAS number : 5888-33-5
EINECS number : 227-561-6
Appearance : Clear colourless fluid liquid
Storage conditions : Room temperature, protected from light

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italy S.p.A., Calco (Lecco), Italy

- Age at study initiation: (P) Males/females: approximately 8 - 9 wks
- Weight at study initiation: (P) Males: 196.5 - 204.7 g; Females: 166.1 - 189.3 g

- Fasting period before study:
- Housing: Pre mating period: no more than 5 per cage in clear polycarbonate cages measuring 59X39X20 cm with a stainless steel
mesh lid and floor (Techniplast - Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray will hold absorbent material which will be
inspected daily and changed at least three times a week.
During mating period: 1 male to one female per cage in clear polycarbonate cages measuring 36X19X24 cm with a stainless steel
mesh lid and floor (Techniplast - Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray will hold absorbent material which will be
inspected daily.
Pregnant females: will be transferred to individual cages after mating: solid bottomed, breeding cages (Techniplast - Gazzada S.a.r.l.,
Buguggiate, Varese), for the gestation period, birth and lactation.
Suitable nesting material will be provided and will be changed as necessary.
- Diet: ad libitum, commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4,
20019, Settimo Milanese (MI), Italy)
- Water: ad libitum, supplied via water bottles

- Acclimation period: aproximately 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2 °C
- Humidity (%): 55 ±15 %
- Air changes (per hr): 15 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: A weighted amount of the test item was dissolved in the vehicle (distilled water) and brought to the final volume appropriate for each concentration. The test solutions were prepared daily at room temperature (concentrations of 5, 10, 20 and 40 mg/ml). All test item concentrations and dosages were based on the test item as supplied (dose volume of 10 ml/kg body weight).
Dose volumes for males were calculated according to individual body weight on the first day of treatment and adjusted according to individual body weight at weekly intervals thereafter. Dose volumes for females were calculated according to individual body weight on the first day of
treatment and adjusted according to individual body weight at weekly intervals up to positive identification of mating. Dose volumes were adjustced to body weight on Days 0, 7, 14 and 20 post-coitum and on Day 0 post-partum. Thereafter individual dose volumes remained constant.
Control animals received the vehicle alone at the same dose volume.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Once during week 1 and week 6 of treatment, samples of prepared formulations were analysed for verification of concentration.

Duration of treatment / exposure:

Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior
to pairing and thereafter through the day before necropsy.
Dose volumes were adjusted once per week for each animal according to the last recorded
body weight.

Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior
to pairing and thereafter during pairing, post coitum and post partum periods until Day 3
post partum or the day before sacrifice.
Dose volumes were adjusted once per week for each animal according to the last recorded
body weight. During the gestation period, dose volumes were calculated according to
individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum.
Thereafter individual dose volumes remained constant.

Frequency of treatment:

once daily

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 male and 10 female per dose group

Control animals:

yes

Details on study design:

Dose levels were selected in consultation with the Sponsor based on information from preliminary study.

Observations and examinations performed and frequency:

Parental observations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
All clinical signs were recorded for individual animals. Examination of individual animals for signs of reaction to treatment was carried out daily
before dosing, immediately after, and approximately 30 minutes and 1 hour after dosing.
Animals were subjected to a detailed clinical examination at weekly intervals. For male animals the clinical signs were performed before treatment
started until necropsy. For female animals clinical signs were performed before treatment started, weekly during the treatment and the mating
periods and on days 0,7, 14 and 20 of gestation and on day 2 post-partum.
Animals were examined in an open arena for a period of three minutes.


BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed at allocation (Study Day -14), at treatment initiation
(Study Day I), and then weekly to pairing. Males were then weighed at weekly intervals up to the day sacrifice, females were weighed on Days 0, 7, 14
and 20 post-coitum and 0/1 and 4 post-partum.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: The weight of food consumed by each cage of rats was recorded weekly following allocation to pairing. Food consumption
for each female was calculated on gestation Days 7, 14 and 20 and on post-partum day 4.

Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: The total litter size (live and dead) was counted alter parturition (Day 0). Live
pups were identified individually within the litter by toe amputation, sexed, and examined for external abnormalities. All pups were examined daily for morbidity and abnormalities dead or abnormal young. All pups were individually weighed on post-partum days 0 and 4. The sexing at birth was
rechecked on Day 4 post-partum.

Sacrifice and pathology:

SACRIFICE
All adult animals were killed with carbon dioxide at the end of the scheduled treatment period (day 31 and day 32 of the study for males and on post partum day 4 for females). Nonpregnant females were killed after Day 25 post-coitum. The number of visible implantation sites were recorded for each dam. Uteri of nonpregnant females were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantatlon.
All pups were killed by intracapular injection of "Tanax".

GROSS NECROPSY
- Gross necropsy: The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surfaces and orifices). Changes were noted and the requisite organs weighed.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs from all animals in all groups were dissected free of fat and weighed:
Testes, Epididymides, Ovaries with oviduct, Uterus with cervix, Prostate and seminal vescicles

In addition from at least 5 male and female animals per group the following organs were dissected free of fat and weighed:
Adrenals, Brain, Heart, Kidneys, Liver, Thymus, Spleen
This was a deviation from the protocol which indicated 5 animals per sex only.

Tissues fixed and preserved
From at least 5 male and female animals per group the following tissues listed below were fixed and preserved in 10% buffered formol-saline (except testes and epididymides which were fixed modified Davidson's fluid and preserved in 70% ethyl alcohol)
Abnormalities Lymph nodes - mesenteric
Adrenal glands Ovaries with oviduct
Brain (only for females) Prostate
Caecum Rectum
Colon Sciatic nerve
Duodenum Seminal vescivles
Heart Spleen
Ileum Stomach
Jejunum Testes
*Kidneys Thymus (where present)
*Liver Thyroid gland
Lungs Trachea
Lymph nodes - cervical Urinary bladder
Uterus with cervix
Vagina
This was a deviation from the protocol which indicated only 5 males and 5 females.

Histopathological examination

After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer
thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test if n was more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The criteria for statistical significance were p<0.05 and p<0.01. The mean value, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

no mortality; clinical signs: salivation

Mortality:

mortality observed, treatment-related

Description (incidence):

no mortality; clinical signs: salivation

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

high proteinuria

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

moderate decrease the relative and absolute thymus weight

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

glandular stomach

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

glandular stomach; thymus

Histopathological findings: neoplastic:

not examined

Details on results:

Mortality and fate of females
No mortality occurred throughout the study.
All females proved to be pregnant.
Total litter loss was observed in 2 high dose females on Days 0 and 2 post partum.
The number of females with live pups on Day 4 post partum was 10 in each of the control,
low and mid-dose groups and 8 in the high dose group.

Clinical observations (Functional Observation Battery Tests)
Clinical observations for neurotoxicity assessment (removal of animals from the home cage
and open arena) did not reveal changes attributable to the test item.

Clinical signs
Salivation was the most relevant clinical sign detected in males and females of the mid- and
high dose group.

Body weight and body weight gain
No relevant difference in body weight and body weight gain was recorded in male animals
throughout the study.
Slight decrease in body weight and body weight gain was recorded in high dose females
during post coitum and post partum periods.

Food consumption
No effects on food consumption were observed in males.
Slight reduction was observed in high-dose females on Day 20 post coitum.

Motor activity and sensory reaction to stimuli
No effects of toxicological significance were noted in all parameters investigated between
control and treated groups.

Haematology
No changes of toxicological significance were recorded.

Clinical chemistry
The main relevant finding was the increase of urea in most of the animals dosed with 500
mg/kg/day (approximately 40%).
The other changes were considered of no toxicological importance.

Urinalyses
High proteinuria was recorded in males dosed with 100 and 500 mg/kg/day, with a doserelated
trend.

Oestrous cycle, reproductive parameters, pairing combination and mating
performance
Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show
intergroup differences.

Implantation, pre-birth loss data and gestation length of females
A slight decrease in the number of corpora lutea and implantation sites was detected in all
treated groups. In addition, a statistically significant reduction was recorded in the total litter
size at birth in the high dose group.
No significant differences were observed in pre-implantation and pre-birth loss.
All dams gave birth between Days 21 and 23 post coitum.

Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups
Statistically significant reduced litter size and litter weight were found in the high dose group
compared to controls, starting from birth up to Day 4 post partum.
In addition, the percentage of cumulative pup loss on Day 4 post partum, starting from the
total litter size of birth, was increased in the high dose group. No differences were found in sex ratio between the groups, both at birth and on Day 4 post partum.

Clinical signs of pups
Most of the clinical signs of pups were comparable between treated and control groups.
An increase number of pups were found dead in the high dose group.

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum
No relevant differences were recorded in decedent pups between the groups.
No relevant findings were observed in pups sacrificed at term.

Terminal body weight and organ weights
At statistical analysis the following findings were observed with respect to the controls:
- slight decrease in terminal body weight in high dose females.
- slight increase in the relative kidney weight of mid- and high dose males.
- moderate decrease the relative and absolute thymus weight of high dose females.

Macroscopic observations
Treatment-related changes were noted in the glandular stomach of both sexes and in the
thymus of the females only.
In the glandular stomach of a single male, treated with the low dose, of a single male and a
single female treated with the mid- dose, and of two males and a single female treated with
the high dose, single or multiple depressed and/or thickened and/or dark areas were noted.
These lesions seen in the mid- and high dose animals correlated histologically with the
presence of single erosions.
In 4/10 and 7/10 females, respectively from the mid- and high dose, the thymus was reduced
in size. Only in 1/10 and 2/10 animals, respectively from the mid- and high dose, this
macroscopic change correlated with histological change.

Microscopic observations
Treatment-related findings were seen in the glandular stomach of both sexes and in the
thymus of the females only.
Stomach (glandular): in a single male and a single female, treated with the mid- dose and in
two males and a single female treated with the high dose, single erosion was seen in the
mucosa.
Thymus: in 1/10 and 2/10 animals, respectively from the mid- and high female dose groups,
different degrees of atrophy of the cortex were seen.
Spermatogenic cycle
Evaluation of the spermatogenic cycle did not show differences between the groups. Regular
layering in the germinal epithelium was noted.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Critical effects observed:

no

Conclusions:

In this OECD TG 422 GLP study, the toxic effects on Sprague Dawley rats of both sexes after repeated dosing with Isobornylacrylate, as well as any effects of the test item on male and female reproductive function such as gonadal function, mating behaviour, conception, parturition and development of offspring up to Day 4 post partum were evaluated.
There was an increase of urea in most of the animals dosed with 500 mg/kg/day (approximately 40%). Statistically significant reduced litter size and litter weight were found in the high dose group compared to controls, starting from birth up to Day 4 post partum. In addition, the percentage of cumulative pup loss on Day 4 post partum, starting from the total litter size of birth, was increased in the high dose group. On the basis of these results the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity was determined to 100 mg/kg/day for males and females.

Executive summary:

In this OECD TG 422 GLP study, both potential repeated dose toxicity and potential reproduction / developmental toxicity of Isobornyl acrylate in Sprague Dawley rats was evaluated.

 

Isobornyl acrylate was administered orally, by gavage, at the dosages of 25, 100 and 500 mg/kg/day. The treatment schedule included 2 weeks before pairing, during pairing, postcoitum and post partum periods up to Day 3 post partum. Animals were administered for approximately 5 and 6 weeks for males and females, respectively.

The parental animals were monitored for daily clinical signs, weekly neurotoxicity assessment, body weight, food consumption and clinical pathology investigations. The dams were allowed to give birth and rear their offspring until Day 4 post partum.

The following results were obtained in the study:

Daily clinical signs: salivation was the relevant sign detected in both sexes of the mid- and high dose groups.

Clinical pathology: no findings of toxicological significance were recorded in the haematological investigation. At clinical chemistry evaluation, increased values of urea were noted in the high dose group of both sexes. At urinalyses high proteinuria was recorded in mid- and high dose males, with a dose related trend.

Reproductive / developmental parameters: no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired up to sperm positive day), the copulatory index (calculated as the percentage of animals mated respect to those paired) and the fertility index, these last two parameters with percentages of 100% in all groups.

All pregnant females in the groups, including controls, had comparable length of gestation period and gave births. Furthermore, sex ratio calculated as the percentage of males in the litter did not differ between the groups.

However, significant differences were found in terms of total litter size and litter weight in the high dose group compared to controls, from birth to Day 4 post partum.

In addition, the percentage of cumulative pup loss on Day 4 post partum, starting from the total litter size at birth, was increased in the high dose group.

No relevant findings were detected in pups sacrificed at term, nor in those found dead in all groups.

Organ weights:increased kidneys weight in mid- and high dose males and a decrease of thymus weight in high dose females were found.

Macroscopic and microscopic observations: treatment-related changes were found in

stomach of mid- and high dose animals of both sexes and in the thymus of mid- and high dose females only.

 

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction / developmental toxicity was determined to100 mg/kg/day for males and females.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

 

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Route of administration:

oral: drinking water

Vehicle:

water

Duration of treatment / exposure:

daily

Frequency of treatment:

13 weeks

Dose / conc.:

0.015 other: % in drinking water

Remarks:

corresponds to approx. 12 mg/kg bw/d in males and 15 mg/kg bw/d in females

Dose / conc.:

0.09 other: % in drinking water

Remarks:

corresponds to approx. 73 mg/kg bw/d in males and 91 mg/kg bw/d in females

Dose / conc.:

0.15 other: % in drinking water

Remarks:

corresponds to approx. 84 mg/kg bw/d in males and 111 mg/kg bw/d in females

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEL

Effect level:

84 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Male rats given the highest dose level had slightly decreased body weight gain but this was considered as not-adverse effect

Remarks on result:

other: no adverse effect observed

Dose descriptor:

NOAEL

Effect level:

111 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

other: no adverse effect observed

Critical effects observed:

no

Conclusions:

In a 13 week oral (drinking water) study similar to OECD TG 408 with butyl acrylate in rats, the NOAEL for males was 84 mg/kg/d and for females 111 mg/kg/d.

Executive summary:

In a 13-week study similar to OECD TG 408, fifteen male and female rats per dose group were exposed to butyl acrylate via drinking water at doses of approximately 12, 73 and 84 mg/kg body weight per day for males and 15, 91 and 111 mg/kg body weight per day for females.Consumption of water was slightly decreased to a similar degree at all concentrations in males and females, respectively. This finding is observed in numerous studies and can be associated with the unpalatability of the test substance. Male rats given the highest dose level had slightly decreased body weight gain. No morphological changes were seen upon light microscopic examination of tissues.

NOAEL (males): 84 mg/kg/d

NOAEL (females): 111 mg/kg/d

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Higher number of test animals: 30 animals (15 m, 15, f) instead of 20 animal (10 m/10 f) per dose group in OECD 408; deviations of the list of microscopic examined organs

Principles of method if other than guideline:

Higher number of test animals: 30 animals (15 m, 15, f) instead of 20 animal (10 m/10 f) per dose group in OECD 408
Deviations of the list of microscopic examined organs

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL.
- Name of test material: Isobornyl Acetate
- Supplier: Bush Boake Allen Ltd., London
- Purity: min. 97%

Species:

rat

Strain:

other: CFE

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not mentioned
- Age at study initiation: weanling rats
- Weight at study initiation: 100-120 g (male); 90-105 g (female)
- Housing: 5 per cage
- Diet: Spiller's Laboratory Small Animal Diet; ad libitum
- Water: ad libitum
- Acclimation period: not mentioned

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-1
- Humidity (%): 50-60
- Air changes (per hr): not mentioned
- Photoperiod (hrs dark / hrs light): not mentioned

Route of administration:

oral: gavage

Details on route of administration:

Gastric intubation
Solution of isobornyl acetate in corn oil
Dosage volume: 5 ml/kg/day

Vehicle:

corn oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks/oral gavage

Frequency of treatment:

daily

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

90 mg/kg bw/day (nominal)

Dose / conc.:

270 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15 males and 15 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

IBA was administered by gastric intubation daily for 13 wk at dose levels of 0, 15, 90 or 270 mg/kg/day to groups of 15 male (body weight 100-120 g) and 15 female (body weight 90-105 g) rats. In addition groups of five rats of each sex received daily doses of 0, 90 or 270 mg/kg/day for 2 to 6 wk. The IBA was diluted in corn oil so that the dosage volume for all rats was 5 ml/kg/day.
The body weight of the animals and their intake of food and water were measured weekly.
Urine was collected from all rats during the final week of treatment and, additionally, during wk 6 from five rats of each sex of each group of the main study. lt was examined for appearance, microscopic constituents and content of glucose, ketones, bile salts and blood. A concentration and dilution
test was carried out on the same rats. At wk 2 this was limited to measurement of the volume and specific gravity of urine produced during a 6-hr
period of water deprivation. At wk 6 and 13 similar measurements were made on the urine produced in 2 hr after a water load of 25 ml/kg andin a 4-hr period after 16 hr without water.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: weely

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinsation: weekly

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes, 24 h fasted before necropsy
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes, 24 h fasted
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: all ratls: during the final week of treatment and 5 rats/sex/dose: during week 6 of the main study
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified


OTHER:

Sacrifice and pathology:

After receiving their final dose of IBA, the animals were fasted for 24 hr and then killed by an overdose of barbiturate.
Blood was collected from the aorta for haematological examination and serum analyses. The blood was examined for haemoglobin content, packed cell volume, counts of erythrocytes, reticulocytes and total and individual types of leucocyte. The serum was analysed for urea (Marsh, Fingerhut & Miller, 1965), glucose (Marks, 1959), total protein (Weichselbaum, 1946) and albumin (Nishi & Rhodes, 1966) and the activities of glutamic-oxalacetic and glutamic-pyruvic transaminase (Karmen, 1955) and lactic dehydrogenase (Wr6blewski & LaDue, 1955) enzymes.
The animals were examined for gross abnormalities and the brain, pituitary, thyroid, heart, liver, spieen, adrenals, kidneys and gonads were weighed. In addition, after 13 wk of treatment the stomach, small intestine and caecum were weighed. Samples of these organs and of Jung, lymph nodes, thymus, urinary bladder, colon, rectum, pancreas, uterus and muscle were preserved in 10 % buffered formalin. Paraffin wax sections of these
tissues were stained with haematoxylin and eosin for microscopic examination.

GROSS PATHOLOGY: Yes
- gross abnormalities were checked

ORGAN WEIGHT:
- brain, pituitary, thyroid, heart, liver, spleen, adrenals, kidneys and gonads were weighed
- stomach, small intestine, caecum were weighed

HISTOPATHOLOGY: Yes
- samples of above mentioned organs were preserved, paraffin section were made and stained and examined microscopically.
- lung, lymph nodes, thymus, urinary bladder, colon, rectum, pancreas, uterus and muscles were also examined microscopically

Statistics:

no data

Clinical signs:

no effects observed

Description (incidence and severity):

No deaths and no abnormalties in behavior and apparance occured during the study.

Mortality:

no mortality observed

Description (incidence):

No deaths and no abnormalties in behavior and apparance occured during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

During the treatment period, there were no significant dfferences in the body weigt or body weight gain observed. A slight decrease in weight in males of the highest dose group became significant after 24h fast prior to death in animals killed at both 6 and 13 weeks.

Dose level Body weight (g) at Week Weight gain (g/rat/day) at Week 13
(mg/kg bw/d) 0 4 8 13
Males
0 108 296 377 439 331
15 107 286 366 424 317
90 107 292 372 429 322
270 107 290 368 420 313

Females
0 99 203 251 283 184
15 101 209 257 293 192
90 99 205 251 279 180
270 99 211 256 289 190

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No differences in food consumption between test and control groups were detected.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was increased in males given 270 mg/kg/d, but not in females.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

After treatment for 6 or 13 weeks, there were no differences in the results for haematological parameters (Haemoglobin, packed cell volume, red blood cells, Reticulocytes, Leucocytes).

Sex and dose level No of rats Hb RBC Leucocytes
Differential (%)
(g/100ml) (10°6/mm³) Total (10³/mm³) N E L M
Males (Week 2)
0 5 12.7 6.13 6.0 11 3 81 5
90 5 13.4 6.66* 6.3 11 4 85 3
270 5 13.4 7.07** 7.5* 14 1 82 3

Females (Week 2)
0 5 12.5 6.47 6.2 4 1 91 4
90 5 13.2 6.38 5.8 8 1 85 6
270 5 13.9** 6.37 7.0 8 2 85 5

Males (Week 6)
0 5 15.7 6.63 10.0 9 0 86 5
90 5 15.7 7.19 10.6 10 1 84 5
270 5 15.4 6.78 10.1 10 2 84 4

Females (Week 6)
0 5 14.9 6.75 7.7 11 2 81 6
90 5 14.0 6.68 5.6 10 2 81 7
270 5 13.7 6.44 7.1 16 2 72 10

Males (Week 13)
0 15 14.9 7.57 8.4 23 2 73 2
15 15 15.0 7.73 8.5 21 1 77 1
90 15 14.5 7.86 9.1 26 1 72 1
270 15 14.8 7.77 8.3 26 1 72 1

Females (Week 13)
0 15 14.1 6.60 6.7 17 1 80 2
15 15 13.8 6.63 6.2 20 1 78 1
90 15 13.9 6.84 5.7 15 1 82 2
270 15 14.0 7.00 5.8 15 2 80 3

HB-Haemoglobin, RBC-recc blood cells, N-Neutrophils, E-Eosinophiles, L-Lymphocytes, M-Monocytes

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The results of serum analysis (GOT, GPT, LDH, glucose, urea, protein, albumin) were similar in test and control rats. For details, see attached publication.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The urine of all rats was of normal colour and free of glucose, bile and ketones.
The females treated with IBA gave results similar to those of control animals.
In males there were no differences between control and IBA-treated rats at wk 2, but at wk 6 cell excretion was increased in rats receiving 270 mg/kg/day and at wk 13 this effect was seen also in animals receiving 90 mg/kg/day. The males given 270 mg/kg/day also showed an impairment of urine-concentrating ability. At wk 6 this was seen only after prolonged (16-20 hr) water deprivation but at wk 13 it was also seen after dehydration for 6 hr. At the lower dosage levels there were no significant changes in the results of the concentration test.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Although the absolute kidney weight was increased only in females at wk 13 the k.idney weight expressed relative to body weight was increased at wk 6 in males and at wk 13 in both sexes. The relative liver weight was increased in both sexes of rats given 270 mg/kg/day for 13 wk. There were no changes in liver weight at wk 2 or 6. Both absolute and relative caecal weights were increased at wk 13 at the highest level of treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities were observed at necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The histopathological examination showed that there was a varying degree of pulmonary change suggestive of a mild infection. This was observed in treated and control animals.
At 270 mg/kg bw-group, an increased incidence of focal tubular degeneration and atrophy and, in males only, a vacuolation of the tubular epithelium. Vacuolation of the epithelial cells of the intrahepatic bile ducts was also seen in males.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY. no effects

BODY WEIGHT AND WEIGHT GAIN: There were no significant differences between test and control animals in the rate of bodyweight gain during the treatment period, but a slight decrease in weight seen in males of the highest dosage group became significant after the 24-hr fast prior to death in animals killed at both 6 and 13 wk.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No differences in food consumption between test and control groups were detected

WATER CONSUMPTION: Throughout the study, water consumption was increased in males given 270 mg/kg/day but not in females.

OPHTHALMOSCOPIC EXAMINATION: no examined

HAEMATOLOGY: After treatment for 6 or 13 wk there were no differences between the test and control groups in the results of the haematological studies. Increases seen at wk 2 in the haemoglobin concentration in female rats and in total leucocyte counts in male rats receiving 270 mg IBA/kg and in erythrocyte counts in males given 90 or 270 mg/kg were not seen at wk 6 or 13. The reticulocyte count of all younger rats was higher than that of
the older animals and red cells showed a marked polychromasia.

CLINICAL CHEMISTRY: The results of the serum analyses were similar in test and control rats

URINALYSIS: The urine of all rats was of normal colour and free from glucose, blood, bile and ketones. The females treated with IBA gave results similar to those of female controls throughout. In males there were no differences between control and IBA-treated rats at wk 2 but at wk 6 cell excretion was increased in rats receiving 270 mg/kg/day and at wk 13 this effect was seen also in animals receiving 90 mg/kg/day. The males given 270 mg/kg/day also showed an impairment of urine-concentrating ability. At wk 6 this was seen only after prolonged (16-20 hr) water deprivation but at wk 13 it was also seen after dehydration for 6 hr. At the lower dosage levels there were no significant changes in the results of the concentration test.

NEUROBEHAVIOUR: no data

ORGAN WEIGHTS: Although the absolute kidney weight was increased only in females at wk 13 the k.idney weight expressed relative to body weight was increased at wk 6 in males and at wk 13 in both sexes (Table 6). The relative liver weight was increased in both sexes of rats given 270 mg/kg/day for 13 wk. There were no changes in liver weight at wk 2 or 6. Both absolute and relative caecal weights were increased at wk 13 at the highest level oftreatment.

GROSS PATHOLOGY:

HISTOPATHOLOGY: NON-NEOPLASTIC: Histological changes associated with IBA treatment were confined to treatment at the 270 mg/kg level. In the kidneys there was an increased incidence of focal tubular degeneration and atrophy and, in males only, a vacuolation of the tubular epithelium. Vacuolation of the epithelial cells of the intrahepatic bile ducts was also seen in males.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): no data

Dose descriptor:

NOAEL

Effect level:

90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: males appear to be more sensitive

Critical effects observed:

yes

Lowest effective dose / conc.:

270 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

no

Conclusions:

In a subchronic oral gavage study with rats the NOAEL was found to be 15 mg/kg/d based on nephrotoxic effects at doses of 270 and 90 mg/kg/d.

Executive summary:

Isobornyl acetate dissolved in corn oil was administered daily to rats by stomach tube in doses of 0 (control), 15, 90 or 270 mg/kg body weight/day for 13 wk. There were no differences between treated and control animals in the rate of body-weight gain, the food intake or the results of haematological investigations. In male rats given 270 mg/kg/day there was a decrease in renal concentrating ability, an increase in water intake, exfoliation of renal tubular cells, increased kidney weight and vacuolation of the renal tubular cells. Signs of nephrotoxicity were also seen with daily doses of 90 mg/kg. Vacuolation of the epithelium of the intrahepatic bile-duct and an increase in liver weights were found at 270 mg/kg. The caeca were also enlarged at this dosage level.

The NOAEL was found to be 90 mg/kg/day.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Groups of 15 Fischer 344 rats/sex/dose were administered acrylic acid in the drinking-water at doses of 83, 250, and 750 mg/kg bw/day over a study period of 3 months.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acrylic acid

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Microbiological Associates, Inc., Walkersville, MD
- Age at study initiation: 41-43 days
- Weight at study initiation: males: 105-142 g; females: 81-110 g
- Housing: 3/4 males or 5 females/cage
- Diet (ad libitum): Zeigler Brothers NIH-07 Rat and Mouse Ration
- Water (ad libitum): tap water

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Fresh solutions were prepared weekly with the concentration of acrylic acid adjusted each time to maintain a relatively constant dosage level.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aqueous solutions of acrylic acid were found to be stable for at least 3 weeks when stored at room temperature using a gas chromatographic procedure. The concentrationof acrylic acid in the dosing preparations was determined monthly.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously

Dose / conc.:

83 mg/kg bw/day (nominal)

Remarks:

in water

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

in water

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

in water

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: none

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION:
- Food consumption for cage determined: Yes, weekly



WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: approx. 2 weeks before sacrifice
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: 15
- Parameters examined: red and white blood cell counts (RBC and WBC), measurement of hemoglobin, calculation of hematocrit and differential and reticulocyte counts.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: approx. 2 weeks before sacrifice
- Animals fasted: Yes
- How many animals: 15
- Parameters examined: measurement of serum concentration of total cholesterol (CHOL), serum urea nitrogen, glucose, alkaline phosphatase (ALP), aspaitate transaminase (AST), alanine transaminase (ALT) and creatine phosphokinase (CPR).


URINALYSIS: Yes
- Time schedule for collection of urine: approx. 2 weeks before sacrifice
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: specific gravity, pH, protein, glucose, ketone, bilirubin, occult blood, nitrite, color and turbidity.


NEUROBEHAVIOURAL EXAMINATION: No
CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly


WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 2 weeks before sacrifice
Rats were fasted for 24 hours prior to bleeding.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 2 weeks before sacrifice
Rats were fasted for 24 hours prior to bleeding.

URINALYSIS: Yes
- Time schedule for collection of urine: 2 weeks before sacrifice
- Metabolism cages used for collection of urine: Yes

Sacrifice and pathology:

Selected tissues examined microscopically from all high dose and control animals included: pituitary, thyroid, parathyroids, adrenals, heart, thymus, spleen, mesenteric lymph node, nasal cavity, trachea, lungs, ovaries and oviducts (or testes and epididymides), kidneys, urinary bladder, tongue, esophagus, stomach, duodenum, colon, liver, pancreas, brain, eyes, skin, manunary gland and sternum. Only organs characterized by gross lesions were examined microscopically from intermediate and low dose animals.

Statistics:

For continuous data, the analysis of variance validated by Bartlett's test, was used. Individual groups were compared with the controls using Duncan's multiple range test. When Bartlett's test indicated heterogeneous variances, individual F tests were used followed by either the Student t-test or the Cochran t-test for unequal variances. Frequency data were compared by the Fisher's exact test Nonparametric data were compared by a distribution-free, multiple comparison method.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in any of the animals.

Mortality:

no mortality observed

Description (incidence):

There were no deaths in the three-month study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In both sexes, mean body weight gain was significantly reduced in the high dose animals throughout the study. At 0,25 mg/kg, weight gain was also reduced in both sexes, though the difference was significant at the end of the study only in the females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significant reductions in diet consumption were observed for both sexes at the high dosage level.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

There was a dose-related reduction in water consumption for both sexes.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

An increase in blood urea nitrogen (BUN) was noted for the high level male rats. In the females, a decrease of serum cholesterol and increases of serum urea nitrogen, glucose, alkaline phosphatase and aspartate transaminase were observed among the high dosage level animals.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In both sexes at the high and intermediate levels, increases of urine specific gravity and urine protein were observed. A decrease in urine pH was noted for the high level female rats as well. Alterations in fluid balance resulting from decreased fluid intake may underlie the changes in serum urea nitrogen, urine specific gravity and urine protein. An alternate explanation of direct or indirect renal toxicity supported by increased relative kidney weights in both sexes at the high and intermediate levels is possible.
In both sexes at the high and intermediate levels, urine specific gravity could not be determined because the refractometer values of most samples were off the scale and the samples were too small for accurate dilution. Urinary protein concentration was significantly increased at the two highest dosage levels. A decrease in urine pH was noted for the high level female rats relative to the controls.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The effect of acrylic acid on the organ weights of the rats was obvious in the high dosage level in both sexes.
In male rats this effect included: (1) Significant reduction in absolute liver, spleen, heart and brain weights; and (2) Significant increase in relative (as % of body weight) liver, kidney, spleen, brain and testes weights.
In the female rats, the effects on the organ weights at the high dose level included: (1) Significant reduction in absolute liver, spleen and heart weights; and (2) Significant increase in absolute kidney weight and relative kidney and brain weights.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicological significant changes observed other than a reduction in water consumption in males

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Critical effects observed:

no

Summary of results of 90 days of inclusion of Acrylic acid in the drinking water of male rats:

 

Dosage goal [mg/kg bw/d]	750		250		83		control
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Dosage attained [mg/kg bw/d]	740	50	250	10	84	4	0	0
Diet consumption [g/kg bw/d]	14.0***	0.6	16.2	0.6	16.4	0.9	16.9	0.6
Water consumption [mL/rat/d]	13.0***	0.7	17.5***	0.8	19.4**	1.3	21.4	0.7
Body weight gain [g]	141.5***	21.5	188.2	23.5	196.7	17.3	200.6	20.2
Liver weight [g]	9.33***	1.25	11.30	1.64	11.17	0.85	11.08	0.89
Liver wt as % bw	3.52*	0.15	3.61	0.38	3.50	0.15	3.40	0.14
Kidney weight [g]	2.06	0.22	2.20	0.18	2.14	0.16	2.15	0.13
Kidney wt as % bw	0.78***	0.04	0.71***	0.03	0.67	0.03	0.66	0.02
Spleen weight [g]	0.53**	0.05	0.60	0.08	0.61	0.06	0.60	0.06
Spleen wt as % bw	0.20**	0.02	0.19	0.02	0.19	0.02	0.18	0.01
Heart weight [g]	0.71**	0.09	0.81	0.10	0.83	0.08	0.82	0.10
Heart wt as % bw	0.27	0.02	0.26	0.02	0.26	0.02	0.25	0.03
Brain weight [g]	1.74**	0.08	1.81	0.08	1.81	0.08	1.84	0.06
Brain wt as % bw	0.66***	0.05	0.58	0.04	0.57	0.03	0.57	0.03
Testes weight [g]	2.72	0.17	2.82	0.14	2.73	0.14	2.72	0.32
Testes wt as % bw	1.04***	0.06	0.91*	0.07	0.86	0.07	0.84	0.11

* p< 0.05, ** p< 0.01, *** p< 0.001

Summary of results of 90 days of inclusion of Acrylic acid in the drinking water of female rats:

 

Dosage goal [mg/kg bw/d]	750		250		83		control
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Dosage attained [mg/kg bw/d]	720	50	250	10	84	3	0	0
Diet consumption [g/kg bw/d]	8.7**	0.3	10.2	0.6	10.6	0.2	10.4	0.4
Water consumption [mL/rat/d]	8.6***	0.5	11.8***	0.7	14.9	0.8	15.0	0.7
Body weight gain [g]	48.0***	8.2	68.4*	9.8	82.1	11.1	78.0	10.8
Liver weight [g]	4.91***	0.50	5.61	0.47	5.84	0.57	5.70	0.45
Liver wt as % bw	3.27	0.22	3.29	0.18	3.16	0.14	3.19	0.13
Kidney weight [g]	1.34**	0.10	1.37***	0.07	1.29	0.08	1.24	0.08
Kidney wt as % bw	0.89***	0.04	0.80***	0.06	0.70	0.04	0.69	0.03
Spleen weight [g]	0.37***	0.04	0.43	0.04	0.46	0.05	0.43	0.05
Spleen wt as % bw	0.24	0.02	0.25	0.02	0.25	0.02	0.24	0.02
Heart weight [g]	0.45***	0.04	0.54	0.05	0.55	0.06	0.54	0.07
Heart wt as % bw	0.30	0.02	0.32	0.02	0.30	0.02	0.30	0.03
Brain weight [g]	1.61	0.12	1.68	0.08	1.66	0.10	1.65	0.14
Brain wt as % bw	1.07***	0.09	0.99	0.08	0.90	0.08	0.93	0.10

* p< 0.05, ** p< 0.01, *** p< 0.001

 

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1 (Guideline study, GLP)

System:

urinary

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity of IBOA has been investigated in a OECD 422 combined repeated dose and reproductive toxicity study.

Existing data gaps are covered using the read across approach based on rapid hydrolysis mentioned in chapter1 of the attached read across justification, considering the ECHA guidance on Read Across (RAAF, ECHA 2017a). For this endpoint, the relatively short half-life of IBOA as parent ester leads to the conclusion that systemic effects after repeated exposure are mainly driven by the products of ester hydrolysis, namely Acrylic Acid, AA, and Isoborneol, IBO. Local effects after inhalation are determined by local release of AA. Butyl Acrylate, BA, and Isobornyl Acetate, IBOAc, are known to hydrolyse also rapidly so that these substances serve as donor substances for the primary metabolites AA and IBO.Qualitatively, this aspect can be categorized as “(Bio) transformation to common compound(s)”. The read across approach is done with a high level of confidence.

Target substance

IBOA

In an OECD TG 422 GLP study, both potential repeated dose toxicity and potential reproduction/ developmental toxicity of IBOA in Sprague Dawley rats was evaluated. IBOA was administered orally, by gavage, at the dosages of 25, 100 and 500 mg/kg/day. The treatment schedule included 2 weeks before pairing, during pairing, postcoitum and post partum periods up to Day 3post partum. Animals were administered for approximately 5 and 6 weeks for males and females, respectively.

The parental animals were monitored for daily clinical signs, weekly neurotoxicity assessment, body weight, food consumption and clinical pathology investigations. The dams were allowed to give birth and rear their offspring until Day 4 post partum.

There was an increase of urea in most of the animals dosed with 500 mg/kg/day (approximately 40%). Statistically significant reduced litter size and litter weight were found in the high dose group compared to controls, starting from birth up to Day 4 post partum. In addition, the percentage of cumulative pup loss on Day 4 post partum, starting from the total litter size of birth, was increased in the high dose group. On the basis of these results the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity was determined to 100 mg/kg/day for males and females. 

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity was determined to100 mg/kg/day for males and females (RTC, 2012).

 

Metabolites and Metabolite Donor Substances

AA

In a well described pre-guideline subchronic study, AA was incorporated in the drinking water of Fischer 344 rats (15 per group) for three months at dosage levels of 750, 250, 83 or 0 mg/kg/day. No deaths occurred during the study but several treatment-related effects were observed. At 750 mg/kg/day dosage level, body weight gain was reduced as were food and water consumption. Changes in organ weights and clinical chemistry parameters were observed along with increases in urine specific gravity and protein concentration, Similar, though less profound, changes occurred at 250 mg/kg/day. At 83 mg/kg/day, the only effect was a reduction of water consumption by male rats. There were no significant treatment-related histopathologic changes. Many of the effects observed may have been the result of decreased water and food consumption rather than specific toxic effects of AA. On the basis of the results obtained in the study, the NOAEL for systemic toxicity was 83 mg/kg bw/d (DePass et al. 1983).

 

In a 12-month study comparable to OECD 452, “Wistar rats (20 rats/group/sex) were given drinking water containing 120, 800, 2000 or 5000 ppm AA (providing doses of about 9, 61, 140 and 331 mg/kg body weight/day, respectively). Satellite groups (10 rats/group/sex) were treated concurrently for 3 months. AA at 5000 ppm, and temporarily also at 2000 ppm, led to reduced drinking water consumption in male rats and, to a slight extent, also in female rats. In the males, feed consumption was reduced at 5000 ppm and body weight gain was retarded at 5000 ppm and marginally also at 2000 ppm. These findings indicate palatability problems and their consequences. There were no indications of systemic toxicity and/or any carcinogenic potential.” On the basis of the results obtained in the study, the NOAEL for systemic toxicity was 61 mg/kg bw/d (Hellwig et al. 1993).

 

BA

In a 13-week study similar to OECD TG 408, fifteen male and female rats per dose group were exposed to butyl acrylate via drinking water at doses of approximately 12, 73 and 84 mg/kg body weight per day for males and 15, 91 and 111 mg/kg body weight per day for females.Consumption of water was slightly decreased to a similar degree at all concentrations in males and females, respectively. This finding is observed in numerous studies and can be associated with the unpalatability of the test substance. Male rats given the highest dose level had slightly decreased body weight gain. No morphological changes were seen upon light microscopic examination of tissues. Nhe NOAEL for male rats was 84 mg/kg/d and for female rats 111 mg/kg/d (Gorzinski et al. 1982).

 

IBOAc

In a 13-week study similar to OECD TG 408,“IBOAc dissolved in com oil was administered daily to rats by stomach tube in doses of 0 (control), 15, 90 or 270 mg/kg body weight/day for 13 wk. There were no differences between treated and control animals in the rate of body-weight gain, the food intake or the results of haematological investigations. In male rats given 270 mg/kg/day there was a decrease in renal concentrating ability, an increase in water intake, exfoliation of renal tubular cells, increased kidney weight and vacuolation of the renal tubular cells. Signs of nephrotoxicity (increased cell excretion) were also seen with daily doses of 90 mg/kg. Vacuolation of the epithelium of the intrahepatic bile-duct and an increase in liver weights were found at 270 mg/kg. The caeca were also enlarged at this dosage level. The NOAEL found was 15 mg/kg/day” (Gaunt Consuelo et al. 1970).

Summary

Systemic toxicity of AA is limited to non-specific toxicity, i.e. effects on body weight gain etc. because of the reduced palatability, with no specific target organ effects other than organ weight increases unsupported by corresponding changes in histopathology. This observation is also made in the study with BA as donor substance for AA. In contrast, specific hepatobilar effects were seen in IBOAc at lower levels as the unspecific effects of AA on molar basis. Specific effects indicative for the hepatobilar system show up also in the study with IBOA (increased urea in the blood). This effect patterns seen in IBO and IBOAC can thus be linked with and assigned to the alcohol metabolite IBO. As a consequence, the alcohol metabolite IBO is assumed to be the toxicological relevant metabolite of IBOA.

Justification for classification or non-classification

According to the criteria as of directive 1272/2008/EC und UN GHS, no classification is warranted.