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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Acceptable, well-documented study which meets basic scientific principles. In this robust study summary results of male animals are reported. In the same study 13 females per each of the dosage groups were treated also. The results of the female animals are reported under IUCLID-Section 7.8.1 (toxicity to reproduction) and 7.8.2 (Developmental toxicity).
Cross-reference
Reason / purpose for cross-reference:
reference to other 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:
daily administration by stomach tube for 42 days.
13 animals per each sex and dose were treated (whereas OECD 422 recommends that each group is started with at least 10 animals of each sex)
GLP compliance:
not specified
Remarks:
but well documented study

Test material

Constituent 1
Chemical structure
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:
Sprague-Dawley
Details on species / strain selection:
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
- 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


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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral 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
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)
dissection ca. 24 hours after the last administration
females: In the same study 13 females per each of the dosage groups were treated also all in all between 38 to 52 days depending on the mating period. The results of the female animals are reported under IUCLID-Section 7.8.1 (toxicity to reproduction) and 7.8.2 (Developmental toxicity).
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40, 200, 1000 mg/kg body weight (mg/kg bw)
Basis:
other: Doses are based on the weekly measured body weights
No. of animals per sex per dose:
13 males
In the same study 13 females per each of the dosage groups were treated also all in all between 38 to 52 days depending on the mating period. The results of the female animals are reported under IUCLID-Section 7.8.1 (toxicity to reproduction) and 7.8.2 (Developmental toxicity).
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: randomly grouped
- Section schedule rationale: all animals were sacrificed
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on administration days 1, 8, 15, 22, 29, 36, 42 and on day of autopsy

FOOD CONSUMPTION : on administration days 1, 8, 29, 36, 42
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to autopsy
- Anaesthetic used for blood collection: Yes - pentobarbital anethesia
- Animals fasted: Yes , for ca. 24 hours
- How many animals: 13 per dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: following blood collection for hematology
- Animals fasted: Yes , for ca. 24 hours
- How many animals: 13 per dose

URINALYSIS: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes :The weights of liver, kidneys, lungs, thymus, testes, epididymis

HISTOPATHOLOGY: Yes: high dose and control animals: liver, heart, thymus, kidney, urinary bladder, lung, spleen, testis and epididymis, altered organs from all other animals (none found)
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%.

Results and discussion

Results of examinations

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
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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
CLINICAL SIGNS:
- 40 mg/kg bw/day:
no differences to the control animals observable
- 200 mg/kg bw:
salivation: 1 animal once,
abnormal respiratory sounds: 2 animals 1-2 times
- 1000 mg/kg bw:
salivation: 13 animals 3-34 times,
rhinorhea: 2 animals 1-2 times,
abnormal respiratory sounds: 5 animals 1 time,

MORTALITY: no

BODY WEIGHT GAIN: 1000 mg/kg bw: reduced

FOOD CONSUMPTION: no statistical differences

FOOD EFFICIENCY: not calculated

WATER CONSUMPTION: not examined

OPHTHALMOSCOPIC EXAMINATION: not examined

HAEMATOLOGY/ CLINICAL CHEMISTRY:
40 mg/kg bw:
reduced: white blood cell count, glucose (small difference)
increased: ratio of eosinophils
200 mg/kg bw:
reduced: lymphocyte ratio, platelet count, glucose
increased: inorganic phosphorus, calcium
1000 mg/kg bw:
reduced: white blood cell count, lymphocyte ratio, platelet count, total
serum protein, glucose
increased: A/G, GPT, GOT, inorganic phosphorus

Degree of variation in platelet count and acidophil ratio was within the
range of physiological variations.
Inorganic phosphorus and calcium varied within the range of
physiological variation.

URINALYSIS: not examined

NEUROBEHAVIOUR: not examined

ORGAN WEIGHTS: no statistical differences

GROSS PATHOLOGY: no dosage related effects

HISTOPATHOLOGY: NON-NEOPLASTIC: no statistical differences

HISTOPATHOLOGY: NEOPLASTIC: no statistical differences

HISTORICAL CONTROL DATA: not given

OTHER FINDINGS

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
clinical signs
Key result
Dose descriptor:
LOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: clinical signs; haematology
Key result
Dose descriptor:
LOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects on body weight change and food consumption, no histological and morphological changes, no adverse effects on hematological and blood chemical findings

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
pharynx
salivary glands
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
presumably yes

Applicant's summary and conclusion

Conclusions:
NOEL: 40 mg/kg bw.;
200 mg/kg bw: Clinical signs: salivation, abnormal respiratory sounds, Hematology/Clinical chemistry: reduced: lymphocyte count, platelet count;
1000 mg/kg bw: Clinical signs: salivation, rhinorhea, abnormal respiratory sounds, 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,
1000 mg/kg bw.: reduced: white blood cell count and glucose, increased A/G

Overall conclusion:
up to 1000 mg/kg bw.: no adverse effects on body weight change and food consumption, no histological and morphological changes, no adverse effects on hematological and blood chemical findings (As summarised in the OECD SIDS (1999)).
Executive summary:

Daily dosages of 0, 40, 200, and 1000 mg/kg bw. were administered by stomach tube to groups of 13 male rats for 42 days. In the same study 13 females per each of the dosage groups were treated also all in all between 38 to 52 days depending on the mating period. The results of the female animals are reported under IUCLID-Section 7.8.1 (toxicity to reproduction) and 7.8.2 (Developmental toxicity). The test-article was formulated in 0.5%CMC and administered in 5 ml/kg bw..

At a dosis of 40 mg/kg bw. the animals showed no differences to the control animals (NOEL).

200 mg/kg bw. lead to salivation and abnormal respiratory sounds. The lymphocyte ratio and the platelet count were reduced.

1000 mg/kg bw. caused salivation, rhinorhea, abnormal respiratory sounds, reduced body weight gain, white blood cell count, platelet count and total serum protein as well as increased segmented neutrophil ratio and increased activity of GPT, GOT and anorganic phosphorus in the serum, females: lung: inflamatory foci.

As summarised in the OECD SIDS (1999) a NOAEL of 1000 mg/kg bw./day was established: “4-Hydroxybenzoic acid induced rale and temporary salivation (sometimes accompanied by rhinorrhea) at 1,000 mg/kg and slightly at 200 mg/kg. These changes were suggesting the irritation of this chemical to respiratory tract. There were no adverse effects on body weight change and food consumption. At necropsy, no histological and morphological changes were observed. In hematological and blood chemical findings of males, decrease in the percentage of lymphocytes and the blood glucose at 200 mg/kg or more groups and decrease in total protein and increase in A/G ratio, GPT and GOT at 1,000 mg/kg were observed. These changes were significant, but not considered adverse effects. Therefore, NOAEL for systemic toxicity was considered to be 1,000 mg/kg/day.”