4-amino-3-nitrophenol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 May 2004 To 2 June 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-3-nitrophenol

EC Number:

210-236-8

EC Name:

4-amino-3-nitrophenol

Cas Number:

610-81-1

Molecular formula:

C6H6N2O3

IUPAC Name:

4-amino-3-nitrophenol

Test material form:

solid: particulate/powder

Remarks:

Dark red powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch no. 0508916
- Expiration date of the lot/batch: September 2005
- Purity test date: 31 August 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in glass flask, stored at +4°C, protected from light and under nitrgen gas
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: solubility : <1g/L at 25°C ; stability : deviations of formulation were within the acceptable range of ±10% (maximal deviation : 9%)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Before preparation, the vehicle was degassed by sonication for at least 15 minutes and then saturated with nitrogen gas, and kept under nitrogen atmosphere for 15 minutes.
The test item was administered as a suspension in the vehicle. The test item was ground to fine powder using a mortar and pestle, suspended in the vehicle at the concentrations of 1, 4 and 80 mg/mL and then homogenized using a magnetic stirrer.
The test item dosage forms were prepared weekly under nitrogen atmosphere and were stored at +4°C, protected from light (using a glass beaker covered with aluminum foil) and under nitrogen atmosphere until delivery.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: 267g (225 to 316g)
- Fasting period before study: not specified
- Housing: The animals were housed individually in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm)
- Diet (e.g. ad libitum): The animals had free access to A04 C pelleted maintenance diet, batch No. 40227 (SAFE, Villemoisson, Epinay-sur-Orge, France) distributed weekly
- Water (e.g. ad libitum):The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation period:a 5-day acclimation period to the conditions of the study preceded 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):

IN-LIFE DATES: From: 11 may 2004 To: 11 June 2004

Administration / exposure

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Before preparation, the vehicle was degassed by sonication for at least 15 minutes and then saturated with nitrogen gas, and kept under nitrogen atmosphere for 15 minutes.
The test item was administered as a suspension in the vehicle. The test item was ground to fine powder using a mortar and pestle, suspended in the vehicle at the concentrations of 1, 4 and 80 mg/mL and then homogenized using a magnetic stirrer

VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification
- Concentration in vehicle: 1,4 and 80 mg/ml
- Amount of vehicle (if gavage): 5mL/kg/day
- Lot/batch no. (if required): carboxymethylcellulose, batch No. 101K0185, supplied by Sigma

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of treatment, the suitability of the proposed preparation procedure was determined by analysis of the homogeneity, stability and concentration of the dosage forms, which were prepared using this procedure. Deviations of formulation were within the acceptable range of ±10% (maximal deviation : 9%).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0

Duration of treatment / exposure:

during the day 6 to 20 of gestation

Frequency of treatment:

once daily

Duration of test:

until the day 20 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 females were used per dose group

Control animals:

yes

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selection of dose-levels for the present study were selected in agreement with the Sponsor based on the results of a previous study (Toxicol Laboratories Ltd, LRL/13/91, 1991) performed at 100, 250 and 600 mg/kg/day.
- Rationale for animal assignment (if not random): the animals were allocated to the groups, according to a stratification procedure based on body weight, recorded on day 0 p.c., to ensure comparatively similar mean body weights in the groups.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day during the treatment period, at least once a day on other days.
- Cage side observations checked : for mortality or signs of morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From arrival, the animals were 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: The body weight of each female was recorded on days 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.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: examination of the principal thoracic and abdominal organs.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

- Other: Any uterine horns without visible implantation sites 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:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Statistics:

Mean values were compared by one-way analysis of variance and the 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.

Historical control data:

Historical control datas were provided in the report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs were noted at 5 mg/kg/day.
At 20 and 400 mg/kg/day, orange colored urines was noted in all animals. Coloration of extremities and fur was also recorded in all animals given 400 mg/kg/day. These findings, which were considered to be evidence of the renal elimination of the test item-related material, were observed many days during the dosing period and suggested systemic exposure to test item following oral administration.
Furthermore, ptyalism was noted in 3/24 females given 400 mg/kg/day, from days 17 or 19 post-coitum until the last dosing.
The above cited findings were not considered to represent adverse effects.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The fluctuations noted in body weight, body weight gain and net body weight change were minor, not statistically significant and not dose-related, consequently, they were not considered treatment-related.

Food consumption and compound intake (if feeding study):

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

The findings recorded at necropsy were low in incidence and concerned two animals in each of the control, 5 and 20 mg/kg/day groups. As no necropsy findings were noted at the high dose-level, the observed findings were considered to be of spontaneous origin.

Maternal developmental toxicity

Pre- and post-implantation loss:

effects observed, non-treatment-related

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

not examined

Description (incidence and severity):

All females were pregnant with live fetuses except one (E29103) in the group given 20 mg/kg/day.

Details on maternal toxic effects:

At all dose-levels, the number of corpora lutea and implantation sites were similar to controls. The only finding of statistical significance was the higher total pre-implantation loss noted at 5 mg/kg/day mainly due to one female; it was therefore considered to be incidental.
The mean post-implantation loss and the mean number of live fetuses were unaffected by the treatment. No dead fetuses were noted in any group.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

maternal abnormalities

mortality

pre and post implantation loss

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The fetal weight was unaffected by treatment.
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:

no effects observed

Changes in sex ratio:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Except for one fetus with a gastroschisis (as a malformation) at 20 mg/kg/day and another fetus with a malrotated paw (as a variation) at 400 mg/kg/day, no malformations or variations were noted. These isolated findings were considered to be unrelated to treatment.

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no soft tissue malformations in any group.
The nature and incidence of the observed soft tissue variations were minor, they were randomly distributed, sometimes with a higher incidence in controls than in treated groups. They were consequently considered not to be treatment-related.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

 

 

 

 

 

Table: 1

 

CLINICAL SIGNS (Summary table/Females/Pregnancy period)

 

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Dose:(mg/kg/day)            0             5             20              400

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Mortality

FINAL SACRIFICE                            24               24               24               24

 

Secretion/Excretion
ORANGE COLOURED URINE	0	0	24	24
PTYALISM	0	0	0	3

 

Abscess/Nodosities

NODOSITIES ON HEAD                           1             0             0             1

ORANGE COLOURED EXTREMITIES 0 0 0 24 ORANGE COLOURED FUR 0 0 0 23

Coloration

Normal

NOREMARKABLEOBSERVATIONS                  23               24                0             0

 

 

 

Table 2 BODY WEIGHT (Mean values/grams/Females/Pregnancy period)

 

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Dose:(mg/kg/day)            0             5             20              400

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DAY 0	MEAN	222 d	224	226	226
	S.D.	19	18	16	15
	N	24	24	23	24
DAY 3	MEAN	245 d	248	249	249
	S.D.	21	19	15	17
	N	24	24	23	24
DAY 6	MEAN	266 d	267	268	269
	S.D.	21	19	17	19
	N	24	24	23	24
DAY 9	MEAN	281 d	281	283	281
	S.D.	22	20	17	19
	N	24	24	23	24
DAY 12	MEAN	300 d	300	303	300
	S.D.	24	22	18	21
	N	24	24	23	24
DAY 15	MEAN	323 d	319	323	320
	S.D.	25	23	20	23
	N	24	24	23	24
DAY 18	MEAN	362 d	361	363	360
	S.D.	29	29	22	28
	N	24	24	23	24
DAY 20	MEAN	396 d	394	399	395
	S.D.	35	31	23	30
	N	24	24	23	24

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Statisticalkey:d=ANOVA+Dunnett-test

 

Table: 3

 

 

 

 

 

HYSTERECTOMY DATA (Summary table)

 

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Dose:(mg/kg/day)            0             5             20              400

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PregnantFemalesAliveatTerm	N	24	24	23	24
with Total Resorptions	N	0	0	0	0
withall Dead Fetuses	N	0	0	0	0
with Live Fetuses	N	24	24	23	24
Corpora Lutea	TOTAL	367	376	342	367
No. peranimal	MEAN	15.3 d	15.7	14.9	15.3
	S.D.	1.9	2.2	2.2	2.3
Implantation Sites	TOTAL	354	348	322	348
No. peranimal	MEAN	14.8 d	14.5	14.0	14.5
	S.D.	2.1	2.1	2.0	2.1
Preimplantation Loss	TOTAL	13 f	28*	20	19
	%	3.5	7.4	5.8	5.2
Fetuses	N	337	333	310	335
No. peranimal	MEAN	14.0 d	13.9	13.5	14.0
	S.D.	2.3	2.4	2.2	2.3
Alive	%	100.0	100.0	100.0	100.0
Dead	%	0.0	0.0	0.0	0.0
Live Fetuses	N	337 f	333	310	335
%of implantation sites		95.2	95.7	96.3	96.3
No. peranimal	MEAN	14.0 d	13.9	13.5	14.0
	S.D.	2.3	2.4	2.2	2.3
Dead Fetuses	N	0 f	0	0	0
%of implantation sites		0.0	0.0	0.0	0.0
No. peranimal	MEAN	0.0	0.0	0.0	0.0
	S.D.	0.0	0.0	0.0	0.0

 

Resorptions+Scars	N	17 f	15	12	13
% of implantation site		4.8	4.3	3.7	3.7
No. peranimal	MEAN	0.7 d	0.6	0.5	0.5
	S.D.	1.0	0.9	0.7	1.3

 

ImplantScars              N             0f            0             0             0

%ofimplantationsites                0.0              0.0				0.0	0.0
No. peranimal	MEAN	0.0	0.0	0.0	0.0
	S.D.	0.0	0.0	0.0	0.0
Resorptions: early	N	17 f	15	12	13
%ofimplantationsites		4.8	4.3	3.7	3.7
No. peranimal	MEAN	0.7 d	0.6	0.5	0.5
	S.D.	1.0	0.9	0.7	1.3
Resorptions: late	N	0 f	0	0	0
%of implantation sites                0.0              0.0				0.0	0.0
No. peranimal	MEAN	0.0	0.0	0.0	0.0
	S.D.	0.0	0.0	0.0	0.0
PostimplantationLoss	TOTAL	17 f	15	12	13
%of implantation sites		4.8	4.3	3.7	3.7
No. peranimal	MEAN	0.7 d	0.6	0.5	0.5
	S.D.	1.0	0.9	0.7	1.3
Male Fetuses	N	163 f	148	144	157
	%	48.4	44.4	46.5	46.9
Female Fetuses	N	174 f	185	166	178
	%	51.6	55.6	53.5	53.1
Fetal Body Weight (g)	MEAN	3.73 d	3.72	3.81	3.66
	S.D.	0.19	0.30	0.18	0.22
Male Fetuses	MEAN	3.83 d	3.83	3.91	3.75
	S.D.	0.21	0.28	0.18	0.24
Female Fetuses	MEAN	3.63 d	3.64	3.71	3.58
	S.D.	0.18	0.29	0.20	0.21

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Statisticalkey:d=ANOVA+Dunnett-testf=Fishersexacttest

 

Table 4 Incidence of fetal skeletal variations (%), litter incidence (%) and mean number of affected fetuses (%)

 

Dose-levels (mg/kg/day)	0	5	20	400
Incomplete ossification of supra occipital bone
.fetal incidence (%)	0.6 [4.2]	1.2 [8.3]	0.6 [4.3]	5.2 [16.7]
.mean affected fetuses/litter (%)	0.6	1.1	0.5	4.9
Incomplete ossification of Hyoid
.fetal incidence (%)	3.5 [16.7]	3.5 [12.5]	9.9 [34.8]	10.3 [37.5]
.mean affected fetuses/litter (%)	3.4	3.2	9.5	9.6
Unossified Hyoid
.fetal incidence(%)	6.4[20.8]	5.2 [12.5]	3.7[26.1]	1.7 [12.5]
. mean affected fetuses/litter	5.9	5.1	3.6	1.7
Unossified 6^thsternebra
.fetalincidence (%)	0.6 [4.2]	1.7 [12.5]	1.2 [8.7]	5.2 [25]
.meanaffected fetuses/litter (%)	0.5	2.1	1.3	5.9
Unossified4^thmetacarpal bone
.fetal incidence (%)	3.5 [20.8]	6.4 [33.3]	1.2 [8.7]	9.2 [25]
.mean affected fetuses/litter (%)	3.4	7.0	1.1	9.7
Short supernumerary 14^thribs
.fetalincidence (%)	0.6[4.2]	1.2[8.3]	1.9[13.0]	4.0[25.0]
.meanaffected fetuses/litter (%)	3.4	3.8	5.2	7.4

[ ]: % of litters affected, *: p<0.05.

HCD: Historical Control Data.

HCD for incomplete ossification of hyoid: mean affected fetuses/litter: minimum= 0, maximum= 19.9%. HCD forunossified6^thsternebra: mean affected fetuses/litter: minimum= 0, maximum= 14.1%.

HCD for unossified 4^thmetacarpal bone: mean affected fetuses/litter: minimum= 2.8, maximum= 41.1%. HCD for incomplete ossification of supraoccipital: mean affected fetuses/litter: minimum= 0, maximum=7.2%.

HCD for short supernumerary 14^thribs: mean affected fetuses/litter: minimum=0.0 ,maximum= 7.6%.

 

 

Applicant's summary and conclusion

Conclusions:

Under the experimental condition of the study, the test item, 4-amino-3-nitrophenol (B051), batch No. 0508916, administered daily by gavage to pregnant female Sprague-Dawley rats from day 6 to 19 post-coitum was well tolerated at 5, 20 and 400 mg/kg/day.
Consequently, the No Observed Adverse Effect Level (NOAEL) for maternal and prenatal toxicity is 400 mg/kg/day.

Executive summary:

The objective of this GLP compliant study was to evaluate the potential toxic effects of the test item, 4-amino-3-nitrophenol (B051), on the pregnant female and on embryonic and fetal development following  daily  oral  administration  (gavage)   to  pregnant   female   rats,   from  days   6   to 19 post-coitum inclusive.

Pregnant SD rats were exposed to the 4 -amino-3 -nitrophenol daily using oral gavage administration at 0, 5, 20, and 400 mg/kg/day from day 6 through day 19 of gestation. Test Item was administered in 0.5% aqueous carboxymethylcellulose.Maternal evaluations and measurements included daily clinical signs, food intake and body weight gain. The dams were sacrificed and subjected to macroscopic examinations, gravid uterus weights were measured and foetuses removed. Typical litter parameters were recorded and foetuses were sexed, weighed and examined. Half of the foetuses were examined for soft tissue anomalies and the other half of the foetuses were examined for skeletal anomalies.

No deaths were reported and clinical signs were limited to orange coloured urine. This fact indicates a renal elimination of test article or its coloured metabolites and thus systemic exposure following oral exposure . An increase of short supernumerary rib was reported at 400 mg/kg/day in foetuses of some of the litters which was however, not statistically significant. Because the incidence (4.1%) was within the historical control range (0.0 – 7.6%) and because no foetuses had full supernumerary rib or abnormal pre-sacral vertebrae, the observation was considered not to be adverse.

Under the experimental condition of the study, the test item, 4-amino-3-nitrophenol (B051), batch No. 0508916, administered daily by gavage to  pregnant female  Sprague-Dawley rats from day 6 to 19 post-coitum was  well  tolerated  at  5, 20 and 400 mg/kg/day. Consequently, the No Observed Adverse Effect Level (NOAEL) for maternal and prenatal toxicity is 400 mg/kg/day.