Isopropyl 4-hydroxybenzoate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL was considered to be 62.5 mg/kg bw for when Sprague-Dawley female rats were treated with isopropyl 4-hydroxybenzoate orally by gavage for 20 days.

Link to relevant study records

Reference

Endpoint:

fertility, other

Remarks:

Estrogenicity

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

Justification for type of information:

Data is from peer-reviewed journal

Qualifier:

according to guideline

Guideline:

other: uterotrophic bioassay

Principles of method if other than guideline:

To study Estrogenic effect of isopropyl 4-hydroxybenzoate in female rats

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): Isopropylparaben (isopropyl 4-hydroxybenzoate)
- Molecular formula (if other than submission substance): C10H12O3
- Molecular weight (if other than submission substance): 180.202 g/mole
- Substance type: Organic
- Physical state: Solid

Species:

rat

Strain:

Sprague-Dawley

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: 21 days
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: Animals were housed in polycarbonate cages
- Diet (e.g. ad libitum): soy-free pellet food
- 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

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: isopropylparaben was administrated in Corn oil

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 and1000 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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: No data available
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Vaginal plugs and/or sperm presence in the vaginal smear was taken as gestation day 0 (GD 0).

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

20 days

Frequency of treatment:

Daily

Details on study schedule:

No data available

Remarks:

0, 62.5, 250 and1000 mg/kg BW/day

No. of animals per sex per dose:

Total: 50
0 mg/kg BW/day: 10 Female
62.5 mg/kg BW/day: 10 Female
250 mg/kg BW/day: 10 Female
1000 mg/kg BW/day: 10 Female
EE (mg/kg BW/day) (Positive control) : 10 Female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data available
- Rationale for animal assignment (if not random): prepubertal female rats were selected.
- Other:doses were adjusted daily for weight changes.

Positive control:

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

Parental animals: Observations and examinations:

No data available

Oestrous cyclicity (parental animals):

proestrous, estrous, metestrous, and diestrous, were determined

Sperm parameters (parental animals):

No data available

Litter observations:

No data available

Postmortem examinations (parental animals):

Changes in body weight, clinical signs and/or abnormal behavior, Clinical chemistry, organ weight and Histopathology were examined.

Postmortem examinations (offspring):

No data available

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. The occurrence of differences in histopathological changes, i.e., decrease of corpora lutea, increase in the number of cystic follicles or decrease of corpora lutea, increase in the number of cystic follicles, was verified using Fisher’s exact probability test between VE and EE, or paraben groups. The rejection value for the null hypothesis was p≤0.05.

Reproductive indices:

No data available

Offspring viability indices:

No data available

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

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:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Dose descriptor:

other: not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Remarks on result:

other: not specified

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

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:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

not specified

Dose descriptor:

other: not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Remarks on result:

other: not specified

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

effects observed, non-treatment-related

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

Sexual maturation:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Body weight: No significant change in body weight was observed in treated rat as compared to control.

Clinical chemistry:
When treated with 250 mg/kg bw, significant decrease in tetra-iodothyronine (T4) level were observed as compared to control.
When treated with 1000 mg/kg bw, significant decrease in Estradiol level were observed as compared to control.

Sexual maturation:
Vaginal opening (VO) day: When treated with 250 and 1000 mg/kg bw, significant delay in Vaginal opening (VO) day were observed as compared to control.
Estrous cycles: When treated with 1000 mg/kg bw, significant decrease in n the number of 4-day estrous cycles were observed as compared to control.

Organ weight:
When treated with 1000 mg/kg bw, significant decrease in ovary and Kidney weight were observed as compared to control.

Histopathology:
When treated with 1000 mg/kg bw, decrease of corpora lutea and increase number of cystic follicles, myometrial hypertrophy of uterus, were observed as compared to control.

Other effect:
Estrogen receptor competitive binding assay: 50% inhibition of ligand binding (IC50) was observed in treated female rats.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

62.5 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

sexual maturation

body weight and weight gain

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

other: No effect

Remarks on result:

other: non toxic

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

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:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

other: not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Remarks on result:

other: not specified

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Reproductive effects observed:

not specified

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Measurement of vaginal opening (VO) day.

Chemical	Dose (mg/kg BW/day)
	62.5	250	1000
VE		33.6 ± 3.23
Isopropylparaben	35.2 ± 3.26	36.2 ± 1.03^a	36.7 ± 0.71^a

a p < 0.05 compared with VE.

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.

Conclusions:

NOAEL was considered to be 62.5 mg/kg bw when Sprague-Dawley female rats were treated with isopropyl 4-hydroxybenzoate orally by gavage for 20 days.

Executive summary:

In a reproductive estrogenicity study, Sprague-Dawley female rats were treated with isopropyl 4-hydroxybenzoate in the concentration of 0, 62.5, 250 and 1000 mg/kg bw from postnatal day 21–40 orally by gavage in corn oil and Ethinylestradiol (1 mg/kg BW/day) was used as a positive control. No significant change in body weight was observed in treated rat as compared to control. Significant decrease in tetra-iodothyronine (T4) level at 250 mg/kg bw and significant decrease in Estradiol level at 1000 mg/kg bw, were observed as compared to control. Similarly, significant delay in Vaginal opening (VO) day were observed at 250 and 1000 mg/kg bw as compared to control. Significant decrease in n the number of 4-day estrous cycles were observed at 1000 mg/kg bw as compared to control. In addition, significant decrease in ovary and Kidney weight were observed at 1000 mg/kg bw as compared to control. 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. Therefore, NOAEL was considered to be 62.5 mg/kg bw when Sprague-Dawley female rats were treated with isopropyl 4-hydroxybenzoate orally by gavage for 20 days.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

62.5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is Klimisch 2 and from peer-reviewed journal

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity: Oral

In a study, isopropyl 4-hydroxybenzoate has been investigated for reproductive toxicity to a greater or lesser extent. The study based on in vivo experiments data in rodents, i.e. most commonly in rats for isopropyl 4-hydroxybenzoate.

In a experimental study conducted by Thuyet al(Reproductive Toxicology 29 (2010) 306–316), Sprague-Dawley female rats were treated with isopropyl 4-hydroxybenzoate in the concentration of 0, 62.5, 250 and 1000 mg/kg bw from postnatal day 21–40 orally by gavage in corn oil and Ethinylestradiol (1 mg/kg BW/day) was used as a positive control. No significant change in body weight was observed in treated rat as compared to control. Significant decrease in tetra-iodothyronine (T4) level at 250 mg/kg bw and significant decrease in Estradiol level at 1000 mg/kg bw, were observed as compared to control. Similarly, reproductive effects such as significant delay in Vaginal opening (VO) day were observed at 250 and 1000 mg/kg bw as compared to control. Significant decrease in n the number of 4-day estrous cycles were observed at 1000 mg/kg bw as compared to control. In addition, significant decrease in ovary and Kidney weight were observed at 1000 mg/kg bw as compared to control. 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. Therefore, NOAEL was considered to be 62.5 mg/kg bw for when Sprague-Dawley female rats were treated with isopropyl 4-hydroxybenzoate orally by gavage for 20 days.

Thus, based on the above studies and predictions on isopropyl 4-hydroxybenzoate, it can be concluded that NOAEL was 62.5 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation, isopropyl 4-hydroxybenzoate can be “Not classified” as reproductive toxicity.

Effects on developmental toxicity

Description of key information

NOAEL was considered to be 62.5 mg/kg bw for F1 generation when Sprague-Dawley female rats were treated with isopropyl 4-hydroxybenzoate orally by gavage for 20 days.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

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

Justification for type of information:

Data is from peer-reviewed journal

Qualifier:

according to guideline

Guideline:

other: uterotrophic bioassay

Principles of method if other than guideline:

To study Estrogenic effect of isopropyl 4-hydroxybenzoate in postnatal female rats

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): Isopropylparaben (isopropyl 4-hydroxybenzoate)
- Molecular formula (if other than submission substance): C10H12O3
- Molecular weight (if other than submission substance): 180.202 g/mole
- Substance type: Organic
- Physical state: Solid

Species:

rat

Strain:

Sprague-Dawley

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: 21 days
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: Animals were housed in polycarbonate cages
- Diet (e.g. ad libitum): soy-free pellet food
- 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

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: isopropylparaben was administrated in Corn oil

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 and1000 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 mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: No data available
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Vaginal plugs and/or sperm presence in the vaginal smear was taken as gestation day 0 (GD 0).

Duration of treatment / exposure:

20 days

Frequency of treatment:

Daily

Duration of test:

21 days (From post natal 21 days to 41 days)

Remarks:

62.5, 250 and 1000 mg/kg bw/day

No. of animals per sex per dose:

Total: 50
0 mg/kg BW/day: 10 Female
62.5 mg/kg BW/day: 10 Female
250 mg/kg BW/day: 10 Female
1000 mg/kg BW/day: 10 Female
EE (mg/kg BW/day) (Positive control) : 10 Female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data available
- Rationale for animal assignment (if not random): prepubertal female rats were selected.
- Other:doses were adjusted daily for weight changes.

Maternal examinations:

not specified

Ovaries and uterine content:

vaginal opening (VO) day and corpora lutea were examined.

Fetal examinations:

Changes in body weight, clinical signs and/or abnormal behavior, Clinical chemistry, organ weight and Histopathology were examined.

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. The occurrence of differences in histopathological changes, i.e., decrease of corpora lutea, increase in the number of cystic follicles or decrease of corpora lutea, increase in the number of cystic follicles, was verified using Fisher’s exact probability test between VE and EE, or paraben groups. The rejection value for the null hypothesis was p≤0.05.

Indices:

not specified

Historical control data:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

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:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Dose descriptor:

other: not specified

Based on:

not specified

Basis for effect level:

other: not specified

Remarks on result:

other: not specified

Abnormalities:

not specified

Localisation:

not specified

Description (incidence and severity):

not specified

Fetal body weight changes:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

not specified

Skeletal malformations:

not specified

Visceral malformations:

not specified

Other effects:

effects observed, treatment-related

Details on embryotoxic / teratogenic effects:

Body weight: No significant change in body weight was observed in treated rat as compared to control.

Clinical chemistry:
When treated with 250 mg/kg bw, significant decrease in tetra-iodothyronine (T4) level were observed as compared to control.
When treated with 1000 mg/kg bw, significant decrease in Estradiol level were observed as compared to control.

Sexual maturation:
Vaginal opening (VO) day: When treated with 250 and 1000 mg/kg bw, significant delay in Vaginal opening (VO) day were observed as compared to control.
Estrous cycles: When treated with 1000 mg/kg bw, significant decrease in n the number of 4-day estrous cycles were observed as compared to control.

Organ weight:
When treated with 1000 mg/kg bw, significant decrease in ovary and Kidney weight were observed as compared to control.

Histopathology:
When treated with 1000 mg/kg bw, decrease of corpora lutea and increase number of cystic follicles, myometrial hypertrophy of uterus, were observed as compared to control.

Other effect:
Estrogen receptor competitive binding assay: 50% inhibition of ligand binding (IC50) was observed in treated female rats.

Dose descriptor:

NOAEL

Effect level:

62.5 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

fetal/pup body weight changes

visceral malformations

other: No effect on chlinical chemistry,

Abnormalities:

not specified

Localisation:

other: not specified

Description (incidence and severity):

not specified

Developmental effects observed:

not specified

Treatment related:

not specified

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Measurement of vaginal opening (VO) day.

Chemical	Dose (mg/kg BW/day)
	62.5	250	1000
VE		33.6 ± 3.23
Isopropylparaben	35.2 ± 3.26	36.2 ± 1.03^a	36.7 ± 0.71^a

a p < 0.05 compared with VE.

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.

Conclusions:

NOAEL was considered to be 62.5 mg/kg bw for F1 generation when Sprague-Dawley female rats were treated with isopropyl 4-hydroxybenzoate orally by gavage for 20 days.

Executive summary:

In a developmental toxicity study, Sprague-Dawley female rats were treated with isopropyl 4-hydroxybenzoate in the concentration of 0, 62.5, 250 and 1000 mg/kg bw from postnatal day 21–40 orally by gavage in corn oil and Ethinylestradiol (1 mg/kg BW/day) was used as a positive control. No significant change in body weight was observed in treated rat as compared to control. Significant decrease in tetra-iodothyronine (T4) level at 250 mg/kg bw and significant decrease in Estradiol level at 1000 mg/kg bw, were observed as compared to control. Similarly, developmental effects such as significant delay in Vaginal opening (VO) day were observed at 250 and 1000 mg/kg bw as compared to control. Significant decrease in n the number of 4-day estrous cycles were observed at 1000 mg/kg bw as compared to control. In addition, significant decrease in ovary and Kidney weight were observed at 1000 mg/kg bw as compared to control. 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. Therefore, NOAEL was considered to be 62.5 mg/kg bw for F1 generation when Sprague-Dawley female rats were treated with isopropyl 4-hydroxybenzoate orally by gavage for 20 days.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

62.5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is Klimisch 2 and from peer-reviewed journal

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity:

In a study, isopropyl 4-hydroxybenzoate has been investigated for developmental toxicity to a greater or lesser extent. The study based on in vivo experiments data in rodents, i.e. most commonly in rats for isopropyl 4-hydroxybenzoate.

In a experimental study conducted by Thuyet al(Reproductive Toxicology 29 (2010) 306–316), Sprague-Dawley female rats were treated with isopropyl 4-hydroxybenzoate in the concentration of 0, 62.5, 250 and 1000 mg/kg bw from postnatal day 21–40 orally by gavage in corn oil and Ethinylestradiol (1 mg/kg BW/day) was used as a positive control. No significant change in body weight was observed in treated rat as compared to control. Significant decrease in tetra-iodothyronine (T4) level at 250 mg/kg bw and significant decrease in Estradiol level at 1000 mg/kg bw, were observed as compared to control. Similarly, developmental effects such as significant delay in Vaginal opening (VO) day were observed at 250 and 1000 mg/kg bw as compared to control. Significant decrease in n the number of 4-day estrous cycles were observed at 1000 mg/kg bw as compared to control. In addition, significant decrease in ovary and Kidney weight were observed at 1000 mg/kg bw as compared to control. 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. Therefore, NOAEL was considered to be 62.5 mg/kg bw for F1 generation when Sprague-Dawley female rats were treated with isopropyl 4-hydroxybenzoate orally by gavage for 20 days.

Thus, based on the above studies and predictions on isopropyl 4-hydroxybenzoate, it can be concluded that NOAEL was 62.5 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation, isopropyl 4-hydroxybenzoate can be “Not classified” as developmental toxicity.

Justification for classification or non-classification

Based on the above studies and predictions on isopropyl 4-hydroxybenzoate, it can be concluded that NOAEL was 62.5 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation, isopropyl 4-hydroxybenzoate can be “Not classified” as reproductive and developmental toxicity.