4-hydroxybenzoic acid

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study which meets basic scientific principles

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

: duration, number of animals, dosages, administration volume, hematology, clinical chemistry, organ weights, histopathologic examination, neurobehaviour

Principles of method if other than guideline:

13 animals per each sex and dose group were treated (whereas OECD 422 recommends that each group is started with at least 10 animals of each sex)

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxybenzoic acid

EC Number:

202-804-9

EC Name:

4-hydroxybenzoic acid

Cas Number:

99-96-7

Molecular formula:

C7H6O3

IUPAC Name:

4-hydroxybenzoic acid

Details on test material:

- Name of test material: 4-hydroxybenzoic acid
- Physical state: solid, white crystalline powder
- Analytical purity: 99.7%
- Impurities (identity and concentrations): 0.02 (w/w)% salicylic acid, 0.03 (w/w)% 4-hydroxyisophthalic acid
- Lot/batch No.: GI0681
- supplier: Ueno Seikyku Co., LTD.
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

other: Sprague-Dawley Crj:CD, SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Nippon Charles River Co., Ltd., Hino Rearing Center
- Age at purchase: 7 weeks
- weight rage at time of grouping: males 305.3 - 348.0 g, females 206.6 - 233.9 g
- Fasting period before study: no
- Housing: individually in metal cages with mesh floor (22 x 27 x 19 cm³) in a rearing room
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week
- Sex: male, female


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +/- 1
- Humidity (%): 50-65
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE: suspension in water
- Concentration in vehicle: 0.5% Carboxymethyl Cellulose Sodium (CMC), Maruishi Seiyaku Co. Ltd., Production No. 1527),
Japan Pharmacopeia injection-quality water, Hikari Seiyaku Co. Ltd., Production No. 9510AH
- Amount of vehicle: 5 ml/kg

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1 (probably) (male animals: see Endpoint study record in section 7.5.1 "Repeated dose Toxicity")
- Length of cohabitation: until copulation/ up to 14 days
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

males: 42 administrations, (2 weeks before mating, 2 weeks during mating and 2 weeks after mating period)
females: in total up to 52 days: 14 days pre-mating period and up to 14 days mating period (until copulation), through the pregnancy period (assuming 20 days pregnancy) until lactation day 3 after delivery (4 days lactation period)

Frequency of treatment:

daily by stomach tube

Details on study schedule:

- Age at mating of the mated animals in the study: 10 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
actual ingested doses were based on the weekly measured body weights/ body weight at the beginning of the pregnancy
Basis:
actual ingested
0, 40, 200, 1000 mg/kg bw./day

No. of animals per sex per dose:

13

Control animals:

yes

Details on study design:

- Control groups: aqueous solution of 0.5% CMC Na
- Dose selection rationale:
Results of a pilot study with 14-days repeated oral administration to male and female rats:
250 mg/kg bw : salivation, abnormal respiratory sounds, rhinorrhea
females: reduced body weight gain
1000 mg/kg bw: salivation, abnormal respiratory sounds, rhinorrhea, reduced body weight gain,
females: reduced: food consumption
- Rationale for animal assignment: ramdomly grouped
- Section schedule rationale: all animals werde sacrificed

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:
males: on administration days 1,8,15,22,29,36,42 and on the day of autopsy,
females: on administration days 1, 8 and 15, on day 22 for females that did not copulate until day 22, postcopulation females: pregnancy days 0, 7, 14, 20 and postpartum females: on lactation days 0 and 4
(administration day 1 = first day of study),

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight, histopathology of testis and epidymidis

Litter observations:

PARAMETERS EXAMINED
numbers of pups born, delivery index, number of pups alive, birth index, live birth index, pup weight on day 0 of lactation, sex ratio, number of pups alive on day 4 of lactation, viability index, pup weight on day 4 of lactation, general status of pups, observation of external deformities


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities

Postmortem examinations (parental animals):

POST-MORTEM EXAMINATIONS: Yes

SACRIFICE
- Male animals after 42 days: All surviving animals
- Female animals: Sacrifice on lactation day 4 (starting with day 0), on the last day of mating period for females that did not copulate, and on the equivalent of pregnancy day 25 for females for which copulation was confirmedbut which did not deliver.

ORGAN WEIGHTS
- Liver, kidneys, lung, thymus, testes, epididymis.

ORGANS FIXED:
- liver, kidneys, lung, thymus, testes, epididymis, brain, heart, spleen, adrenals, uterus, and bladder

HISTOPATHOLOGY (high dose and control animals):
- Liver, heart, thymus, kidney, urinary bladder, lung, spleen, testis, and epididymis

Postmortem examinations (offspring):

HISTOPATHOLOGY
The organs in the thoracic and abdominal cavities were excised together and were fixed in 10% formalin and preserved according to litter. The carcasses were fixed in ethanol and preserved according to litter.

Statistics:

Mean graded data for histopathological findings were tested for significant differences between the control and various dosage groups using Mann-Whitney's U-test, and positive grade totals using Fisher's direct probability one-tailed test. For all other data, which values obtained for individuals the uniformity of distribution of the various groups was first tested by Bartlett's method. When this resulted in a uniform distribution, a one-dimensional distribution analysis was performed, and when intergroup significance was observed, the differences in the mean values between the control and various dosage groups were tested using Dunnett's method if the number of animals per group was the same, or Scheffé's method when it was not. When the distribution was not uniform or when there were groups for which the distribution was 0, the Kruskal-Wallis rank sum test was performed. When intergroup significance was observed, Dunnett's or Scheffé's method tests the differences between the control and various dosage groups were performed. The level of significance was 5% or 1%.

Reproductive indices:

number of mated pairs, number of copulated pairs, copulation index, number of pregnant animals, fertility index, pairing days until copulation, frequency of vaginal estrus, number of pregnant females, number of pregnant females with pups alive, gestation index, gestation lenth in days, number of corpora lutea, number of imlantation sites, implantation index, number of pups born, delivery index

Offspring viability indices:

number of pups alive, birth index, live birth index, pup weight on day 0 of lactation, sex ratio, number of pups alive on day 4 of lactation, viability index, pup weight on day 4 of lactation, general status of pups, observation of external deformities

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

With doses 200 mg/kg bw. and above abnormal respiratory sounds or transient post-administration salivation

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

suppressed weight gain in males with doses of 1000 mg/kg bw./day

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

With doses 200 mg/kg bw. and above reduced lymophocyte ratio was abserved

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males with doses of 200 mg/kg bw and above reduced glucose was observed.
With dose of 1000 mg/kg bw. reduced total protein and elevated GPT, GOT and increased A/G ratio was observed.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Male Animals:
NOEL: 40 mg/kg bw.
200 mg/kg bw: transient post-administration salivation, abnormal respiratory sounds, Hematology/Clinical chemistry: males reduced lymphocyte ratio and reduced glucose level ,
-1000 mg/kg bw: Clinical signs: salivation, rhinorhea, abnormal respiratory sounds, males reduced: body weight gain, Hematology/Clinical Chemistry: reduced: white blood cell count, platelet count, total serum protein, increased: segmented neutrophil ratio, GPT, GOT, inorganic phosphorus.

The following significant differences were seen as merely statistical effects caused by the biological variation and not based on a test item related biological effect:
40 mg/kg bw.: reduced: glucose and white blood cell count, increased: ratio of eosinophil ,
200 mg/kg bw.: reduced: glucose, increased: blood calcium,
1000mg/kg bw.: reduced: white blood cell count and glucose, increased A/G-40 mg/kg bw: no observable effects

FEMALE ANIMALS:
NOEL: 40 mg/kg bw
-200 mg/kg bw: abnormal respiratory sounds: 3 animals 1-2 times
-1000 mg/kg bw: salivation: 13 animals 2-23 times, rhinorhea: 1 animal 1time, abnormal respiratory sounds: 9 animals 1-10 times, lungs: inflamatory foci (1 in the control group, 4 cases in the dosage group)

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

Effect level:

> 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

2

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

haematology

clinical biochemistry

3

Key result

Dose descriptor:

NOEL

Remarks:

reproductive/developmental toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

reproductive performance

Remarks on result:

other: Generation: P and F1 (migrated information)

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

respiratory system: upper respiratory tract

Organ:

blood

nasal cavity

oral cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

CLINICAL SIGNS (OFFSPRING): no abnormalities in the general status.
BODY WEIGHT (OFFSPRING): no significant differences between the control group and the various dosage groups werde observed in males or females in body weights on day 0 or lactation day 4.

GROSS PATHOLOGY (OFFSPRING): no external deformities were observed in the surviving pups on the day of birth. In autopsies of pups on lactation day 4, no abnormalities were observed in the control, nor were any abnormalities in the autopsy of the dead pup.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

gross pathology

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Daily dosages of 0, 40, 200, and 1000 mg/kg bw./day were administred by stomach tube to groups of 13 male and female rats for a 14 days pre-mating period, for a mating period up to 14 days. The administration to the males was continued for 2 further weeks, totally 42 days. The administration to the females was continued for the pregnancy period of 20 days and for a lactation period of 4 days. All in all between 38 to 52 days (depending on the mating period). The test-article was formulated in 0.5%CMC, the administration volume was 5 ml/kg bw.
NOEL = 40 mg/kg bw.,
NOEL for Repeated dose toxicity: 40 mg/kg bw./day,
NOEL for Reproductive toxicity: 1000 mg/kg bw./day,
NOEL for Developmental toxicity: 1000 mg/kg bw./day,
Pups: NOEL: 1000 mg/kg bw/day.

Executive summary:

Daily dosages of 0, 40, 200, and 1000 mg/kg bw./day were administred by stomach tube to groups of 13 male and female rats for a 14 days pre-mating period, for a mating period up to 14 days. Whereas OECD 422 recommends that each group is started with at least 10 animals of each sex. The administration to the males was continued for 2 further weeks, totally 42 days. The administration to the females was continued for the pregnancy period of 20 days and for a lactation period of 4 days. All in all between 38 to 52 days (depending on the mating period). The test-article was formulated in 0.5%CMC, the administration volume was 5 ml/kg bw.

NOEL = 40 mg/kg bw..

200 mg/kg bw./day lead to abnormal respiratory sounds and additionally with the male animals to salivation and reduced lymphocyte ratio and platelet count.

1000 mg/kg bw: salivation, abnormal respiratory sound, rhinorhea, males: reduced body weight gain, white blood cell count, platelet count, total serum protein, increased: segmented neutrophil ratio, GPT, GOT, inorganic phosphorus, females: lung: inflamatory foci.

- NOEL for Repeated dose toxicity: 40 mg/kg bw./day

- NOEL for Reproductive toxicity: 1000 mg/kg bw./day

- NOEL for Developmental toxicity: 1000 mg/kg bw./day