Isopropyl 4-hydroxybenzoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Potential estrogenic effect(s) of parabens at the prepubertal stage of a postnatal female rat model

Author:

Thuy T.B. Vo, Yeong-Min Yoo, Kyung-Chul Choi, Eui-Bae Jeung

Year:

2010

Bibliographic source:

Reproductive Toxicology, 29 (2010) 306–316

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refere below principle

Principles of method if other than guideline:

Repeated dose oral toxicity study was performed to determine the estrogenic effects of Isopropylparaben on postnatal female rats

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isopropyl 4-hydroxybenzoate

EC Number:

224-069-3

EC Name:

Isopropyl 4-hydroxybenzoate

Cas Number:

4191-73-5

Molecular formula:

C10H12O3

IUPAC Name:

propan-2-yl 4-hydroxybenzoate

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): isopropyl 4-hydroxybenzoate
- Molecular formula : C10H12O3
- Molecular weight : 180.202 g/mole
- Smiles notation : c1(C(OC(C)C)=O)ccc(O)cc1
- InChl : 1S/C10H12O3/c1-7(2)13-10(12)8-3-5-9(11)6-4-8/h3-7,11H,1-2H3
- Substance type:Organic
- Physical state:Solid

Specific details on test material used for the study:

- Name of test material: Isopropyl p-hydroxybenzoate
- IUPAC name: isopropyl 4-hydroxybenzoate
- Molecular formula: C10H12O3
- Molecular weight: 180.202 g/mol
- Substance type: Organic
- Physical state: No data

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

No data

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dea Han Biolink Co., Ltd. (Eumsung, Chungbuk, Korea).
- Age at study initiation: At the time of purchase: F0 Male: 10-week-old F0 Female : 8 week old
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: in polycarbonate cages
- Use of restrainers for preventing ingestion (if dermal): No data available
- Diet (e.g. ad libitum): soy-free pellet food ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%):No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12 h light/12 h dark

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

No data

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in corn oil to give a dose range of 0, 62.5, 250 or 1000 mg/Kg bw/day

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil
- Concentration in vehicle: 0, 62.5, 250 or 1000 mg/Kg bw/day
- Amount of vehicle (if gavage): 5 ml/kg BW/day
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

Duration of treatment : PND 21-40
Duration of exposure: 21 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

0, 62.5, 250 or 1000 mg/kg BW/day

No. of animals per sex per dose:

Total: 50
0 mg/Kg bw: 10 females
62.5 mg/Kg bw: 10 females
250 mg/Kg bw: 10 females
1000 mg/Kg bw: 10 females
Positive controls: 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): prepubertal female rats were selected.
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data

Positive control:

Ethinylestradiol (EE) (1 mg/kg BW/day) were used as positive control.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data available
- Cage side observations checked in table [No.?] were included.: Abnormal behaviour

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No data available

BODY WEIGHT: Yes
- Time schedule for examinations: throughout experimental period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data available
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available
- Time schedule for examinations: No data available

HAEMATOLOGY: No data
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood was collected at 24 h after the final oral treatment on PND 41
- Animals fasted: No data available
- How many animals: All animals
- Parameters checked in table [No.?] were examined. serum concentration of estradiol, prolactin, tetra-iodothyronine and thyroid-stimulating hormone

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined. No data available

NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data available

OTHER: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, The uterus, ovary, livers, kidneys, adrenal glands, pituitary glands, and thyroid glands were removed under ether anesthesia, stripped of fat, and weighed.

HISTOPATHOLOGY: Yes, organs were taken for histopathological tests. The uterus, ovary, livers, kidneys, adrenal glands, pituitary glands, and thyroid glands were fixed in 10% neutral buffered formalin immediately after weighing, then dehydrated at room temperature for 24 h, and embedded in paraffin. The tissues were then sectioned to a thickness of 5µm across the mid-region of each tissue, and mounted on slides. These sections were stained with hematoxylin and eosin (H&E), and the histopathological changes in organ tissues were examined under a light microscope.

Other examinations:

The ability of the test chemical to bind to binding receptors was determined with an ERα and ERβ screening system using a fluorescent estrogen, Fluormone ES2. Absorbances were analyzed on a fluorescence microplate reader. The Fluormone ES2 was excited at 485 nm, and the fluorescence was analyzed at 530 nm. The inhibition rate of binding to receptor ERα or ERβ was calculated from the absorbances.

Statistics:

Data for the vaginal opening (VO) day, bodyweight, and organ weight of the rats were statistically evaluated by one-way ANOVA. Significant differences between treatment groups and the respective controls were tested using Tukey’s multiple regression test at p < 0.05. The variable category of the incidence data in the ovarian changes was used as the outcome.

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Details on results:

Clinical signs and mortality: No data

Body weight and weight gain: No significant change was observed in treatment groups for body weight.

Food consumption and compound intake: No data

Food efficiency: No data

Water consumption and compound intake: No data

Opthalmoscopic examination: No data

Haematology: No data

Clinical chemistry: Estradiol hormone levels were reduced while prolactin levels were increased isopropylparaben, but the changes were not dose dependent. Estradiol hormone levels were significantly reduced by 1000 mg/Kg bw dose. A decrease in T4 concentration was observed at 62.5 mg/Kg bw when compared with the VE control but was not significant. No significant differences in the TSH levels of all treatment groups were observed.

Urinanalysis: No data

Neurobehaviour: No data

Organ weights: No significant change was observed in treatment groups for uterus wet weight and pituitary gland weight. Decrease in ovary weight and kidney weight was observed in 1000 mg/kg BW concentration.

Gross pathology: No data

Histopathology: Histopathological changes in the ovaries and uteri were observed in peripubertal female rats following treatments with parabens. In all treatment groups, decrease of corpora lutea, and an increasing number of cystic follicles, or the thinning of the follicular cells were observed in the ovaries of peripubertal rats. There were significant differences in histopathological changes, i.e. decrease of corpora lutea, increase number of cystic follicles or not decrease of corpora lutea, increase number of cystic follicles, between VE group and 1000 mg/Kg bw isopropylparaben. The uterus showed some effects in the muscle layers, including myometrial hypertrophy, in response to the 1000 mg/Kg bw isopropylparaben. No differences between the control group and any of the treatment groups for the histopathology of thyroid and adrenal glands

Other: The binding affinities isopropylparaben to ERα and ERβ were investigated and the concentration of a chemical giving 50% inhibition of ligand binding (IC50) was determined in vitro. IC50 values for 17β-estradiol binding to ERα and ERβ as positive controls were 2.99×10−9 and 3.03×10−9 M, respectively. Isopropylparaben demonstrated a relative responses of 1.52×10−5 M.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No significant alterations were observed at the mentioned dose level

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Effects of parabens on body weight and organ weight (uterine, pituitary, ovary, adrenal gland, thyroid gland, kidney, liver) in peripubertal female rats.

Groups	Body weight (g)	Uterus weight (mg/g BW)	Pituitary weight (mg/g BW)	Ovary weight (mg/g BW)	Thyroid weight (mg/g BW)	Kidney weight (mg/g BW)	Adrenal weight (mg/g BW)	Liver weight (mg/g BW)
VE	118.69 ± 6.4	1.29 ± 0.11	0.043 ± 0.008	0.51 ± 0.06	0.86 ± 0.07	8.31 ± 0.57	0.27 ± 0.01	42.90 ± 2.17
EE (mg/kg BW/day)	61.56 ± 4.85^b	3.40 ± 0.30^b	0.077 ± 0.012^b	0.45 ± 0.05	1.23 ± 0.1^b	9.94 ± 0.63^b	0.38 ± 0.03^b	52.38 ± 2.77^b
Isopropylparaben (mg/kg BW/day)
62.5	110.67 ± 7.85	1.19 ± 0.30	0.045 ± 0.005	0.41 ± 0.06	0.85 ± 0.07	8.04 ± 0.44	0.29 ± 0.03	41.22 ± 3.00
250	113.85 ± 5.87	1.15 ± 0.44	0.047 ± 0.009	0.04 ± 0.07	0.82 ± 0.06	7.66 ± 0.47	0.27 ± 0.02	40.58 ± 3.19
1000	102.45 ± 4.38	0.87 ± 0.22	0.044 ± 0.003	0.32 ± 0.05^a	0.88 ± 0.08	7.25 ± 0.35^a	0.3 ± 0.02	41.75 ± 2.04

Mean±SD; n = 10 prepubertal female rats/group.

^ap < 0.05 vs. vehicle (VE) (Tukey’s multiple regression test at p < 0.05).

^bp < 0.01 vs. vehicle (VE) (Tukey’s multiple regression test at p < 0.05).

Histopathological effects of parabens on the ovaries and uteri of peripubertal female rats.

Groups	Ovary			Uterus
	Decrease of corpora lutea, increase in the number of cystic follicles (n = 10), n (%)	Not decrease of corpora lutea, increase in the number of cystic follicles (n = 10), n (%)	P-value*	Thickness of morphometric measurement (m)
VE	0 (0%)	10 (100%)		125.64 ± 4.44
EE (mg/kg BW/day)	7 (70%)	3 (30%)	0.0015	253.85 ± 27.74^b
Isopropylparaben (mg/kg BW/day)
62.5	2 (20%)	8 (80%)	0.2368	141.03 ± 8.88
250	3 (30%)	7 (70%)	0.1052	189.74 ± 32.03
1000	5 (50%)	5 (50%)	0.0162	212.82 ± 4.44^a

Mean±SD; 10 rats were examined for each dose.

* Fisher’s exact probability test for category variables (categorized histopathological changes: decrease of corpora lutea, increase in the number of cystic follicles or not decrease of corpora lutea, increase in the number of cystic follicles on the vehicle (VE) and EE or paraben exposure groups) respectively. There rejection value for the null hypothesis was p≤0.05.

^ap < 0.05 vs. vehicle (VE) (Tukey’s multiple regression test at p < 0.05).

^bp < 0.01 vs. vehicle (VE) (Tukey’s multiple regression test at p < 0.05).

Effects of parabens on circulating estradiol, prolactin, and T4 levels in peripubertal female rats.

Groups	Estradiol (pg/ml)	Prolactin (ng/ml)	T4 (ng/ml)
VE	47.07 ± 14.72	9.81 ± 3.12	3.00 ± 0.32
EE (mg/kg BW/day)	55.37 ± 14.77	238.93 ± 35.00^b	2.73 ± 0.50
Isopropylparaben (mg/kg BW/day)
62.5	30.53 ± 15.91	45.10 ± 13.12	2.70 ± 0.39
250	25.20 ± 6.05	49.76 ± 58.31	1.73 ± 0.34^b
1000	16.23 ± 6.86^a	24.12 ± 6.46	3.06 ± 0.19

Mean±SD; n = 10 prepubertal female rats/group.

^ap < 0.05 vs. vehicle (VE) (Tukey’s multiple regression test at p < 0.05).

^bp < 0.01 vs. vehicle (VE) (Tukey’s multiple regression test at p < 0.05).

Relative binding affinities of various parabens to ERαand ERβ.

Compound	ERα		ERβ
	IC50^a	RBA^b	IC50^a	RBA^b
17βEstradiol	2.99×10^−9M	100	3.03×10^−9M	100
Isopropylparaben	1.52×10^−5M	0.019	1.69×10^−5M	0.017

Each value was the mean of triplicate determinations in more than four experiments.

^aIC50 is the concentration of the paraben compound that inhibits binding of Fluormone ES2 to ER by 50%.

^bRBA was calculated by the equation: (IC50 of 17-estradiol/IC50 of parabens)×100.

Applicant's summary and conclusion

Conclusions:

The No Observed adverse effect level for isopropylparaben is found to be 250 mg/Kg bw/day when exposed to female Sprague Dawley rats.

Executive summary:

Subacute repeated dose toxicity study was performed to determine the toxic nature of isopropylparaben. The chemical dissolved in corn oil was given by oral route of expoure to peripubertal female Sprague Dawley animals at dose levels of 0, 62.5, 250 or 1000 mg/kg BW/day daily from postnatal day (PND) 21–40. Ethinylestradiol (1 mg/kg BW/day) was used as a positive control. The animals were observed for changes in body weight, organ weight, serum parameters, gross pathology and histopathology. No significant change in body weight was observed in treated rat as compared to control. Significant decrease in tetra-iodothyronine (T4) level and significant decrease in Estradiol level at 1000 mg/kg bw, were observed as compared to control. No significant change was observed in treatment groups for uterus wet weight and pituitary gland weight. Decrease in ovary weight and kidney weight was observed in 1000 mg/kg BW concentration. Histopathological changes in the ovaries and uteri were observed in peripubertal female rats following treatments with isopropylparaben. The uterus showed some effects in the muscle layers, including myometrial hypertrophy, in response to the 1000 mg/Kg bw isopropylparaben. No differences between the control group and any of the treatment groups for the histopathology of thyroid and adrenal glands. Decrease of corpora lutea and increase number of cystic follicles, myometrial hypertrophy of uterus, were observed at 1000 mg/kg bw as compared to control. 50% inhibition of ligand binding (IC50) was observed in treated female rats. Hence, the The No Observed adverse effect level for isopropylparaben is found to be 250 mg/Kg bw/day when exposed to female Sprague Dawley rats.