3-aminophenol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

The above study has been conducted to establish a relation and to assess and evaluate teratological effects of the test chemical in Sprague Dawley rats.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

3-aminophenol

EC Number:

209-711-2

EC Name:

3-aminophenol

Cas Number:

591-27-5

Molecular formula:

C6H7NO

IUPAC Name:

3-aminophenol

Details on test material:

- Name of test material (as cited in study report): m-Aminophenol
- Molecular formula: C6H7NO
- Molecular weight: 109.12
- Substance type: Organic
- Batch number: 220117
- Physical state: White powder
- Titre: 99%
- Storage conditions: At +4°C, protected from light and under nitrogen gas
- Expiry date: September 2005

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L’Arbresle, France.
- Age at study initiation: 10 weeks old
- Weight at study initiation: Animals had a mean body weight of 263 g (range: 227 g to 306 g).
- Fasting period before study: No data available
- Housing: The animals were housed individually in suspended wire-mesh cages. A metal tray, containing autoclaved sawdust, was placed under each cage. The sawdust was changed at least once a week.
- Diet (e.g. ad libitum): The animals had free access to A04 C pelleted maintenance diet distributed weekly.
- Water (e.g. ad libitum): Tap water (filtered with a 0.22 μm filter).
- Acclimation period: Animals were acclimated to the conditions of the study for 5 days before the beginning of the mating period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): About 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00-19:00)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Before preparation, the vehicle was degassed by sonification for at least 30 minutes. The test chemical was administered as a suspension in the vehicle.
The test chemical was ground to fine powder using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 6, 20 and 60 mg/mL and then homogenized using a magnetic stirrer. The test material dosage forms were prepared under nitrogen atmosphere for up to 9 days and were stored at +4°C, protected from light (using a glass beaker covered with aluminum foil) and under nitrogen atmosphere until delivery.

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):
- Concentration in vehicle: 0.5% methylcellulose solution
- Amount of vehicle (if gavage): 5 mL/kg/day
- Lot/batch no. (if required): 014K0081
- Purity: No data available

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No Data Available

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight
- Proof of pregnancy: Each morning following mating, rats with spermatozoa found in a vaginal smear or sperm plug in situ were considered as pregnant animals. The day where evidence of mating is found was designated as day 0 post-coitum
- Allocation to group: Before day 3 post-coitum., the animals were allocated to the groups, according to a stratification procedure based on body weight, recorded on day 0 post coitum. to ensure comparatively similar mean body weight among groups. A larger number of animals than necessary were paired to permit selection and/or replacement of individuals before start of treatment. The data of the replaced animals are filed but not presented in the study report.
- Identification: Each animal was individually identified by an ear tattoo and received a unique identity number.

Duration of treatment / exposure:

Day 6 to day 19 post-coitum

Frequency of treatment:

Daily

Duration of test:

20 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 100 or 300 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

24 female rats per group. In total 96 female rats were used.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels of the present study were selected in agreement with the Sponsor and based on the preliminary prenatal study in which the test chemical was administered daily by gavage to pregnant Sprague-Dawley rats from days 6 to day 19 post coitum. at the dose-levels of 250, 500 or 1000 mg/kg/day.
All pregnant females treated at 1000 mg/kg/day were prematurely sacrificed on GD 6 or 7 (GD: gestation day) due to toxic signs (tremors and startled behavior). Hypersalivation, lacrimation, staggering gait, tachypnea, piloerection and/or half-closed eyes were also recorded prior to sacrifice. At 500 or 250 mg/kg/day no deaths occurred, however, numerous clinical signs were recorded (hypersalivation, lacrimation, staggering gait, respiratory difficulties and/or half-closed eyes, and hypotonia in group 2 only).
Body weight loss was noted transiently in the 500 mg/kg/day group during GD 6-9. Treatment with the test item induced a reduction in body weight gain and food consumption in a dose-related manner. The net body weight change of females given 500 or 250 mg/kg/day was lower than controls.
None of the litters were affected by treatment with the test item at 250 mg/kg/day. The mean fetal body weight was slightly lower in the 500 mg/kg/day group than in the control group. There were no external malformations or variations in any groups.
Based on these results, the following dose-levels were selected:
. the low dose-level of 30 mg/kg/day was expected to be the No Observed Effect Level,
. the intermediate dose-level of 100 mg/kg/day was expected to produce minor maternal toxicity,
. the high dose-level of 300 mg/kg/day was expected to produce a reasonable level of maternal toxicity.

- Rationale for animal assignment (if not random):
The treatment groups are detailed in the following table:
- Group 1: 24 females mated, treated with 0 mg/kg/day,
- Group 2: 24 females mated, treated with 30 mg/kg/day,
- Group 3: 24 females mated, treated with10 0 mg/kg/day,
- Group 4: 24 females mated, treated with 300 mg/kg/day,

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day before the treatment period and at least twice a day during the treatment period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From arrival, each animal was observed at least once a day as part of routine examinations. From the start of the treatment period, each animal was observed at least once a day, at approximately the same time for the recording of clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: 0, 3, 6, 9, 12, 15, 18 and 20 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The quantity of food consumed by each female was recorded for the following intervals:
. days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20 post-coitum.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: On day 20 post coitum, females were sacrificed by asphyxiation using carbon dioxide followed by cervical dislocation and were submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.

The ovaries and uterus of the females were also examined.

OTHER: No Data Available

Ovaries and uterine content:

-The ovaries and uterine content was examined after termination: Yes

-The weight of the gravid uterus was recorded for each pregnant female (with at least one live fetus).
Examinations included:
. number of corpora lutea,
. number and distribution of dead and live fetuses,
. number and distribution of early and late resorptions,
. number and distribution of uterine scars,
. number and distribution of implantation sites.

-The following classification was used to record:
. uterine scar: uterine implantation without implant,
. early resorption: evidence of implant without recognizable embryo,
. late resorption: dead embryo or fetus with external degenerative changes,
. dead fetus: non live fetus with discernible digits.

-Uterine horns without visible implantation site were immersed in an aqueous solution of ammonium sulphide (Salewski) to reveal the presence of uterine scars.

-A gross evaluation of placentas was also undertaken.

Fetal examinations:

- Body weight: Yes, The body weight of each fetus was recorded.

- External examinations: Yes, Each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices. The fetuses were then killed by a subcutaneous injection of thiopental sodium.

- Soft tissue examinations: Yes, Approximately half of the fetuses in each litter were selected, aiming at limiting imbalanced distribution of sexes, were fixed with Harrison's fluid. A detailed soft tissue examination was performed according to a free-hand serial sectioning technique, which included the observation of all the organs and structures of the head, neck, thorax and abdomen.

- Skeletal examinations: Yes, The remaining fetuses per litter were eviscerated and then fixed with ethyl alcohol. A detailed examination of the skeleton (bone) was performed after staining with alizarin red S (modified Dawson technique). This examination included the observation of all the bone structures of the head, spine, rib cage, pelvis and limbs.

- Sex of live fetuses: Yes, The sex of each fetus was determined at the time of evisceration or at the time of serial sectioning.

Statistics:

Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.

Indices:

Fetal Viability Indices, Implantation Indices, Fetal Indices.

Historical control data:

No Data Available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs
No clinical signs were observed during the study in the control and low-dose group. At 100 mg/kg/day, the urine was colored in orange in 7/23 pregnant and one non-pregnant female animal, generally from GD 10 (GD: gestation day) and limited to a few days. At 300 mg/kg/day, all animals had orange colored urine from GD 13 (generally) until the end of the dosing period in the majority of cases. Excessive salivation was noted in a few animals (4/24), on rare occasions. Colored urine in both groups suggested systemic exposure to the test item. No other remarkable clinical signs were noted during the study, areas of hair loss or abnormal growth of teeth being regularly observed in pregnant rats of this strain.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No premature deaths occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At day 18 and 20 of gestation, the body weight was slightly but statistically lower at 300 mg/kg as compared to controls. This was accompanied by a significant decrease in food consumption from day 6 to 12 and from day 15 to 20 of gestation as compared to controls. The net body weight gain from day 6 of gestation and onwards was significantly lower at 300 mg/kg (mean gain 15 g.) as compared to controls (mean gain 35.7 g.).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The amount of food consumed by the test chemical-treated groups at 30 or 100 mg/kg/day was similar to controls. At 300 mg/kg/day, the amount of food consumed was lower than controls (often statistically significant), throughout the whole dosing period.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy findings
There were no treatment-related findings at the autopsy of the animals. At 300 mg/kg/day, the observation of brownish contents in the vagina of female was not clearly ascribed to the test item-treatment as this finding was observed at a very low incidence.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Significant increase in post-implantation loss at 300 mg/kg compared to control grou.

Total litter losses by resorption:

not specified

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Significant increase in early resorption at 300 mg/kg compared to control group.

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of dead fetuses per dam did not differ significantly.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Pregnancy rate:
A total of 23/24, 22/24, 23/24 and 24/24 females in control, 30, 100 and 300 mg/kg/day groups respectively were pregnant with live fetuses at scheduled sacrifice.

Other effects:

not specified

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

early or late resorptions

effects on pregnancy duration

gross pathology

maternal abnormalities

mortality

pre and post implantation loss

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Average fetal weight of males was higher at 30, 100 and 300 mg/kg compared to controls. Effect not dose-related.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The average number of live fetuses per dam was similar between all groups.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The average number of fetuses per dam was similar between all groups.

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External observations:
No external findings were observed in control and test-material treated groups at 30 or 300 mg/kg/day. At 100 mg/kg/day, two malformed fetuses were observed from a single litter. One fetus displayed atrophy of the head, astomia, arhinia, ablepharia, absence of the eye bulge, microtia and malpositioned pinna. The other showed anal atresia and thread-like tail. Considering the very low incidence of these malformations and in absence of evident dose-relationship, these external findings were considered to have occurred by chance. Therefore they were not ascribed to the test material-treatment.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal observations:
There were no treatment-related malformations of the skeleton of fetuses in any group. There were no treatment-related variations of the skeleton of the fetuses in treated groups at 30 or 100 mg/kg/day. A short supernumerary 14th rib was recorded in half of the litters from the high-dose group

Visceral malformations:

no effects observed

Description (incidence and severity):

Visceral observations:
No findings at visceral examinations of the fetuses were considered to be compound-related.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

other: embryotoxicity

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

Description (incidence and severity):

Observed in half of the litters at 300 mg/kg dose level.

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for embryo-fetal toxicity was considered at 100 mg/kg bw/day when pregnant rats were exposed to the test chemical from day 6 to 19 of gestation.

Executive summary:

In this reproductive and developmental toxicity study by L’Oreal (2004), pregnant Sprague-Dawley rats were exposed to 3-aminophenol by oral gavage at 0, 30, 100 and 300 mg/kg bw/day from day 6-19 of gestation. At day 18 and 20 of gestation, the body weight was slightly but statistically lower at 300 mg/kg as compared to controls. This was accompanied by a significant decrease in food consumption from day 6 to 12 and from day 15 to 20 of gestation as compared to controls. The net body weight gain from day 6 of gestation and onwards was significantly lower at 300 mg/kg (mean gain 15 g.) as compared to controls (mean gain 35.7 g.). The amount of food consumed by the test chemical-treated groups at 30 or 100 mg/kg/day was similar to controls. At 300 mg/kg/day, the amount of food consumed was lower than controls (often statistically significant), throughout the whole dosing period.There was a significant increase in early resorption rate and in post-implantation loss of embryos at 300 mg/kg as compared to controls. The biological significance of these results are doubtful as there was no adverse effect on any other parameter of reproduction, as evident by no significant effect on the number of corpora lutea per dam, the number of implantation sites per dam, the number of fetuses per dam, or the number of live fetuses per dam. There were no dead fetuses produced in any of the treatment groups. The fetal sex ratio was similar between the groups and the average fetal weight of males was higher at 30, 100 and 300 mg/kg as compared to controls (not dose related). A significant increase in rudimentary 14^thribs was observed in fetus/litters from the 300 mg/kg dose group as compared to controls. This effect is of doubtful clinical significance as such sites of ossification are most likely to be absorbed postnatally by the adjacent vertebrae during the lateral transverse processes (J Toxicol. Environ Helath B Crit Rev 2004; 7: 437 -49). Apart from rudimentary 14^thribs at 300 mg/kg, there were no treatment-related malformations or variations observed following the external, soft tissue and skeletal examinations. The No Observed Adverse Effect Level (NOAEL) for embryo-fetal toxicity was considered at 100 mg/kg bw/day when pregnant rats were exposed to the test chemical from day 6 to 19 of gestation.