Isopropyl 4-hydroxybenzoate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

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:

Toxicological evalution of isopropylparaben and isobutyparaben mixture in Sprague-Dawley rats following 28 days of dermal exposure.

Author:

Min Ji Kim, Seung Jun Kwack, Seong Kwang Lim, Yeon Joo Kim,Tae, Hyun Roh, Seul MinChoi,Hyung Sik Kim,Byung Mu Lee

Year:

2015

Bibliographic source:

Regulatory Toxicology and Pharmacology 73(2015) 544-551

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Principles of method if other than guideline:

28 Days repeated dose dermal toxicity study was performed to determine the dermal toxic nature of isopropylparaben using Sprague Dawley 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
- Purity: 99%
- Impurity: 1%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio (Sungnam, South Korea)
- Females (if applicable) nulliparous and non-pregnant: No data
- Age at study initiation: 5 weeks old at purchase
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: The animals were housed in cages in a controlled environment
- Diet (e.g. ad libitum): The animals were fed 5L79 (Charles River rat and mouse 19% Auto) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data

DETAILS OF FOOD AND WATER QUALITY: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 °C
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 h light/dark

IN-LIFE DATES: From: To: No data

Administration / exposure

Type of coverage:

occlusive

Vehicle:

ethanol

Details on exposure:

TEST SITE
- Area of exposure: Dorsal area of the trunk
- % coverage: Not less than 10% of the body surface area
- Type of wrap if used: porous gauze dressing and non irritating tape. The test site was further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals could not ingest the test substance
- Time intervals for shavings or clipplings: Shaving was carried out approximately 24 h before the test and repeat clipping or shaving was performed at approximately weekly
intervals.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No data
- Time after start of exposure: No data

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 µl
- Concentration (if solution): 0, 50, 100, 300 or 600 mg/Kg bw
- Constant volume or concentration used: No data
- For solids, paste formed: Yes, the chemical was appled in cream form

VEHICLE
- Justification for use and choice of vehicle (if other than water): Ethanol
- Amount(s) applied (volume or weight with unit): 100 µl
- Concentration (if solution): No data
- Lot/batch no. (if required): No data
- Purity: No data

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, Restrainers may be used to prevent ingestion of the test substance, but complete immobilisation is not a recommended method.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

28 days

Frequency of treatment:

5 days/week

Doses / concentrations

Remarks:

0, 50, 100, 300 or 600 mg/Kg bw

No. of animals per sex per dose:

Total: 50
0 mg/Kg bw/day: 10 animals
50 mg/Kg bw/day: 10 animals
100 mg/Kg bw/day: 10 animals
300 mg/Kg bw/day: 10 animals
600 mg/Kg bw/day: 10 animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses for animal treatment were selected based on assumptions regarding human exposure to parabens. The total daily use of cosmetics and toiletries containing paraben is assumed to be 10 g/day, based on the reports that about half of the average daily amount of product use (17.4-19.4 g/day) contains parabens as a
preservative. The maximum authorized concentration is 0.4%, which gives the maximal exposure of paraben at 4 mg/g. The calculation for the maximal daily exposure is: (daily exposure) ¼ (amount daily use) X (conc. in product) ÷ (weight of human), which gives approximately 0.7 mg/ kg. Human weight was considered to be 60Kg. The uncertainty factor (UF) was then estimated at 300 [animal to human, human to human, an additional modifying factor for the use of subacute data]. Therefore, the maximal daily level of paraben exposure was observed to 210 mg/kg bw. The maximal daily dosage group in this experiment was determined to be 600 mg/kg bw, which is approximately three times higher than the estimated level of human exposure for a single paraben.

- Rationale for animal assignment (if not random): Yes, before the test, animals are randomized and assigned to the treatment and control groups.

- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data

Examinations

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice Daily

DERMAL IRRITATION (if dermal study): No data available
- Time schedule for examinations: No data available

BODY WEIGHT: Yes
- Time schedule for examinations: Twice a week throughout the experiment

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, twice a week throughout the experiment

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 data available

WATER CONSUMPTION: Yes
- Time schedule for examinations: Twice a week throughout the experiment

OPHTHALMOSCOPIC EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to necrospy
- Anaesthetic used for blood collection: Yes (Diethyl ether)
- Animals fasted: Yes, overnight fasting was done
- How many animals: All animals
- Parameters checked in table [No.?] were examined.; Hemoglobin, hematocrit, red blood cell, white blood cell, platelet, mean corpuscular volume, mean corpuscular volume concentration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to necrospy
- Animals fasted:Yes, overnight fasting was done
- How many animals: All animals
- Parameters checked in table [No.?] were examined.- albumin, glucose, total cholesterol, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenous, blood urea nitrogen, creatinine, sodium, potassium, chloride, calcium, phosphorus, triglyceride, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase and gamma glutamyltranspeptidase. Hormone levels, Triiodothyronine (T3), follicle-stimulating hormone (FSH), and estradiol concentrations, insulin levels, the concentration of testosterone and thyroid-stimulating hormone (TSH) were determined

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: Hormone level ( T3 ,Follical stimulating hormones, and estradiol) was measured by ELISA Test.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, Liver, kidneys, heart, brains, testes, and prostates from male rats, and ovaries and vaginas from female animals were removed and weighed. The dorsal skin from the site of application was also removed from animals.

HISTOPATHOLOGY: Yes, Brains, hearts, kidneys, the biggest lobe of livers, and sectioned dorsal skin were fixed in a solution of 10% neutral formalin. The tissues were post-fixed and embedded in paraffin. Three to 5 mm thick sections were cut and stained with hematoxyline and eosin (H&E) for light microscopy.

Statistics:

All data were statically analyzed with SPSS®12.0 KO for Windows (SPSS Inc.), using one-way ANOVA with Tukey's test followed by Duncan's test.

Results and discussion

Results of examinations

Clinical signs:

not specified

Dermal irritation:

not specified

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):

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

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:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

Clinical signs and mortality:
Mortality: No death of animals occurred during experiment.

Clinical signs: No data available

Dermal Irritation: No data available

Body weight and weight gain: Average body weight was found similar in all groups dosed 0, 50, 100, 300 or 600 mg/Kg bw

Food consumption and compound intake: Food consumption was found to be similar in all groups dosed 0, 50, 100, 300 or 600 mg/Kg bw

Food efficiency: No data available

Water consumption and compound intake: Water consumption was found to be similar in all groups dosed 0, 50, 100, 300 or 600 mg/Kg bw

Opthalmoscopic examination: No data available

Haematology : No significant effects were observed

Clinical chemistry: There were no significant differences in serum T3, TSH, insulin, E2, or testosterone concentrations between control and isopropylparaben-treated groups of female rats. There ere no statistical differences in FSH level in male rats between control and test animals of the isopropylparaben treated group

Urinanalysis: No data available

Neurobehaviour: No data available

Organ weights: The relative weight of heart and kidneys increased in a dose dependent manner by simultaneous application of the test chemical in male rats. However, significant difference in organ weights were not observed in female rats. The vagina and uterus, as a target organs for endocrine disruptors, did not show any significant change.

Gross pathology: No data available

Histopathology: No significant histopathological signs were observed in the brain, liver, heart, and kidney, although some lesions which can naturally occur were found. All IPP-treated groups did not show any specific lesions in either male or female rats.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean relative organ weight of male rats treated in the skin with isopropyl paraben

 

Groups	Brain (g)	Heart (g)	Liver (g)	Prostate (g)	Kidney (L)	Kidney (R)	Testis (L)	Testis (R)
0	2.025 ± 0.138	1.131 ± 0.075	12.446 ± 1.095	0.453 ± 0.071	1.157 ± 0.046	1.160 ± 0.056	1.559 ± 0.123	1.527 ± 0.117
50	2.076 ± 0.053	1.161 ± 0.049	12.788 ± 0.954	0.462 ± 0.067	1.178 ± 0.036	1.163 ± 0.053	1.566 ± 0.166	1.543 ± 0.165
100	2.046 ± 0.118	1.148 ± 0.051	12.746 ± 0.806	0.443 ± 0.041	1.168 ± 0.056	1.173 ± 0.036	1.561 ± 0.124	1.539 ± 0.124
300	2.066 ± 0.135	1.150 ± 0.078	12.630 ± 1.562	0.453 ± 0.088	1.172 ± 0.052	1.165 ± 0.069	1.559 ± 0.175	1.546 ± 0.161
600	2.086 ± 0.170	1.166 ± 0.059	12.523 ± 0.678	0.460 ± 0.090	1.156 ± 0.045	1.173 ± 0.103	1.591 ± 0.150	1.575 ± 0.154

 

Table 2: Serum hormone concentrations of male SpragueeDawley rats treated with isopropyl paraben

Groups	T3 (ng/mL)	TSH (ng/mL)	Insulin (ng/mL)	E2 (pg/mL)	FSH (IU/L)	Testosterone (ng/mL)
0	0.78 ± 0.06	2.09 ± 0.55	1.57 ± 0.44	15.41 ± 3.29	4.54 ± 0.60	3.88 ± 0.58
50	0.81 ± 0.06	2.19 ± 0.70	1.61 ± 0.35	14.75 ± 2.34	3.61 ± 0.57	3.81 ± 0.61
100	0.76 ± 0.04	2.01 ± 0.48	1.62 ± 0.37	16.03 ± 3.16	3.48 ± 0.45	3.79 ± 0.50
300	0.80 ± 0.06	1.81 ± 0.38	1.65 ± 0.44	15.60 ± 2.95	3.36 ± 0.56	3.61 ± 0.67
600	0.77 ± 0.05	1.61 ± 0.52	1.39 ± 0.51	13.49 ± 2.69	3.29 ± 0.79	3.48 ± 0.77

 

Table 3: Histopathology of skin of female SD rats treated with isopropyl paraben, isobutyl paraben or mixture of two parabens.

Control group	Control group
No specific lesion	3
Isopropyl paraben (IPP) group	50	100	300	600
No specific lesion	3	3	3	3
Inflammation, dermis, (multi)focal	0	0	0	0
Minimal	0	0	0	0

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect level (NOAEL) for isopropyl paraben in male and female Sprague Dawley rats is 600 mg/Kg bw/day.

Executive summary:

28 Days repeated dose dermal toxicity study was performed to determine the dermal toxic nature of isopropylparaben using male and female Sprague Dawley rats. The study was performed as per OECD guideline 410. The test chemical was dissolved in 99% pure ethanolat dose levels of 0, 50, 100, 300 or 600 mg/Kg bw/day. Shortly before testing, fur was clipped from the dorsal area of the trunk of the test animals. Shaving was carried out approximately 24 h before the test and repeat clipping or shaving was performed at approximately weekly intervals. Not less than 10 per cent of the body surface area was cleared for the application of the test substance. Groups of 10 male and 10 female rats were topically applied IPP dissolved in 100 ml of ethanol (99%), 5 days per week for 28 days. The test substances were applied to the shaved dorsal skin of the animals. Each subject was administered the cream at 50 µl/cm2 of body surface area. Between applications the test substance is held in contact with the skin with a porous gauze dressing and non irritating tape. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Animals were observed twice daily; clinical findings and body weights were recorded. Changes in body weight and the amounts of food or water consumption were recorded throughout the experiment. Brains, hearts, kidneys, the biggest lobe of livers, and sectioned dorsal skin were fixed in a solution of 10% neutral formalin and observed through light microscopy. No mortality was noted during the study. Average body weight, food and water consumption were similar among all groups. The relative weight of heart and kidneys increased in a dose dependent manner by simultaneous application of IPP in male rats. Significant difference (p < 0.05) in relative weight of kidney and testis was observed in male. There was no significant difference in hematological evaluation. There were no significant differences in serum T3, TSH, insulin, E2, or testosterone concentrations between control and isoparaben-treated groups of female rats. There ere no statistical differences in FSH level in male rats between control and test animals. The vagina and uterus, as a target organs for endocrine disruptors, did not show any significant change among groups. No significant histopathological signs were observed in the brain, liver, heart, and kidney, although some lesions which can naturally occur were found. All IPP-treated groups did not show any specific lesions in either male or female rats. Based on the observations made, the No Observed Adverse Effect level (NOAEL) for isopropyl paraben in male and female Sprague Dawley rats is 600 mg/Kg bw/day.