4-tert-butylphenol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991/08/28 - 1991/11/27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted May 12, 1981

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: 88/302/EEC

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nonylphenol

EC Number:

246-672-0

EC Name:

Nonylphenol

Cas Number:

25154-52-3

IUPAC Name:

2-nonylphenol

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Versuchstierzucht, 8741 Sulzfeld, Germany
- Age at study initiation: females about 8 weeks
- Weight at study initiation: a) 183 to 369 g (groups I (a), II, III; b) 203 to 269 g (groups I (b), V)
- Fasting period before study: no data
- Housing: Makrolon cages, cleaned twice a week
- Diet (e.g. ad libitum): as libidum, "Ssniff R" pelleted diet (Alleindiät für Ratten)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: a) 7 days, b) 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 °C +- 1,5 °C
- Humidity (%): rel. humidity: 50 to 70 %,
- Air changes (per hr): 16 times per hour, (filtered adequately)
- Photoperiod (hrs dark / hrs light): artificial light (120 lux) from 7.00 a.m to 7.00 p.m.


IN-LIFE DATES: 20 days (gestation day 0 - 20)
date of receipt: a) August 21, 1991; b) October 16, 1991

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Prepared freshly each day according to study specific SOP


VEHICLE
- Justification for use and choice of vehicle (if other than water): according test substance data sheet
- Concentration in vehicle: 0 g/l, 15 g/l, 30 g/l, 60 g/l
- Amount of vehicle (if gavage): 5 ml/kg b.w.
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC Analysis of nonylphenol.
The test article was analysed by reverse phase HPLC in corn oil samples. The samples were taken from dosing solutions prepared for the teratogenicity study in Wistar rats, treated orally. The analysed concentration values of the samples show good accordance with the nominal values.

Details on mating procedure:

- Impregnation procedure: co-housed
- If co-housed:
- M/F ratio per cage: 1:2
- Length of cohabitation overnight:
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear and / or vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

6-15 day of gestation

Frequency of treatment:

Daily per oral administration, adjusted daily according to the weight development of the animals

Duration of test:

20 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 75, 150, 300 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

groups number of females animal no. daily dose (mg/kg b.w.) volume of administration concentration (g(l)
I (a) 32 151-182 0 5 0
I (b) 25 101-125 0 5 0
V 25 551-575 75 5 15
II 25 251-275 150 5 30
III 32 351-382 300 5 60

vehicle: corn oil

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: preliminary dose range finding IBR project no.: 20-04-0502/01-91
- Rationale for animal assignment (if not random): random
- Justification for route of administration: oral in order to obtain high resorption rates and is recommended by OECD guideline

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during the entire period of the investigation.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the entire period of the investigation.
With regard to: sensory and motor behaviour, coat, urine and fecal excretion, conditions of body orifices.
Viability: checked twice a day
Mortality was recorded on a daily basis.
Additionally, dose response examinations were carried out in appropriate intervals after administration of the test article during treatment.
During gestation females were observed closely for signs of abortion or premature delivery.


BODY WEIGHT: Yes
- Time schedule for examinations: daily during the dosing period
- recorded only at the beginning of the study (day 0) and additionally at days 6, 10, 15, 20 of gestation
- body weight development was evaluated over the following phase: 0-6, 6-15, 15-20 and 0-20


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- Food consumption for each animal determined and evaluated for body weight development: Yes
- Food consumption was determined by re-weighting non-consumed diet at the end of each weighting interval.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: reproductive organs


OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- weights of fetuses and placenta: Yes
- Number of corpora lutea: No data
- Number of implantations and locations: Yes
- Number and location of live and dead fetuses: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (visceral effects)
(Ref. Wilson, J.G. (1965) Methods for administering agents and detecting malformations in experimental animals
in: J.G. Wilson and J. Warkany (eds) Teratology, Principles and Techniques, Univ. of Chicago Press, Chicago, London, pp. 262-277)
- Skeletal examinations: Yes: half per litter
(Ref. Dawson, A.B. (1926), A note on the staining of the skeleton of cleaned specimens with alizarin red S, Strain Technology 1, 123-124
- Head examinations: No data

Statistics:

Analyses of Variance with a subsequent multiple range test for growth (body weight changes and food consumption) and reproduction parameters (numbers of fetuses, resorptions, implantations, corpora lutea and weights of fetuses, placentae and uteri) or if indicated
group mean values were compared by the "Kruskal-Wallis test" and "Mann-Whitney U-test".
The maternal weight on day 20 of gestation was corrected for gravid uterine weight and the corrected rate of body weight gain (day (0-20 of gestation) was calculated.
Statistiical evaluation of dose groups II and III was done relative to control group I (a), and dose group V relative to control group I (b)

Indices:

abortion rate, resorption rate, pre-implantation loss index, post-implantation loss index, live birth index, runts index, variation index, anomaly index, malformation index

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical observation:
- Salvation was noted after treatment of all dose groups
- An increased discharge of urine was found in all animals of the high dose group by estimation of the degree of moisture in the bedding. The onset of this sign was predominantly at day 8 of gestation.

2. Abortion rate
- No females showed signs of abortion or premature delivery in the course of the study.

3. Body weight development
- Body weight gain of group III females was significantly reduces during treatment during the whole study period. Both, the total and the corrected weight gains were affected from day 0 - 20 of gestation.
- Weight gains of group II and V did not differ significantly compared either to group I (A) nor group I (b).

4. Food consumption
In correspondence to the weight gain, food consumption of group III females was significantly decreased between days 6-15 and 0-20 compared to group I (a)

5. Necroscopy findings
A slight dose relationship may be found in the findings of kidneys and spleen in group II and III.
Five females of group III and one female of group II had pale kidneys, 2 group III females showed irregularly shaped kidneys. Two females of group III and one of group II showed reddening of renal pelvis. In 2 group III females and one of group II, the spleen appeared reduced size.
In group IV, treatment with 600 mg/kg was stopped due to the high mortality of animals, pale kidneys (16 of 18 animals) and spleens with reduces size (17 of 18 animals) were found. In 4 animals irregularly shaped kidneys were noted and in 8 animals a reddening of medulla or pelvis.
These findings seem to elucidate the above described dose relationship.
The effect on the kidneys seems to correspond to the increased discharge of urine which was observed during clinical observations in group III.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

mortality

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- The mean number of fetuses did not differ significantly between control and treated groups.
- The mean number of fetuses in the left or right uterine horns did not differ significantly between control and treated groups.
- No biological significant differences were determined among groups concerning the number of fetuses with head or tail presentation
- The sex ration of male and female fetuses did not differ significantly between control and treated groups.
- The mean fetal body weight did not differ significantly between control and treated groups (irrespective of sex).
- The mean placental weights did not differ significantly between control and treated groups.
- No runts were found in the dose groups.
- There were no dead fetuses in any group.
- The number of resorptions did not differ significantly between control and treated groups.
- Resorption index and post implantation index did not differ significantly between control and treated groups.
- The Pre-implantation loss index did not differ in a clearly dose-related manner.
- The mean numbers of implantation did not differ significantly between control and treated groups.
- No relevant differences were found in the left/right intrauterine distribution
- The total number of corpora lutea did not differ significantly between control and treated groups.
- Respective numbers with regard to the left and right ovary did not differ significantly between control and treated groups.

- One malfunction was found in group I (b) and one minor abnormality was found in each of the groups I (a) and V. These findings are considered to be incidental
- Skeletal examination: One malformed fetus was found in each of the control groups.
The incidence of skeletal abnormalities did not reveal any evidence of a treatment effect.
Incomplete ossification of the skull was observed in 76 % of the litters and in 41 % of the fetuses of group III, which seemed slightly increased compared to group I (a). However, historical data show a mean index of 80,1 % of litters and 47,6 % of fetuses. => no indication of treatment related differences.
- no abnormalities were found during visceral examination of fetuses.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall toxicity

Fetal abnormalities

Key result

Abnormalities:

not specified

Overall developmental toxicity

Key result

Developmental effects observed:

not specified

Any other information on results incl. tables

		Group
		Ib	V	I	II	III
1.    Caesarean Section
a. Fetuses
Number of fetuses	mean	15.0	14.0	15.1	15.3	13.5
	S.D.	2.3	2.4	4.1	2.0	3.8
Sex Ratio (m/f)		1.0	1.0	1.3	0.9	1.1
Weights of fetuses (g) (Males / Females)	mean	3.91	3.94	3.92	3.96	3.98
	S.D.	0.20	0.30	0.31	0.44	0.48
Weights of fetuses (g) (Males)	mean	4.03	4.01	4.02	4.08	4.11
	S.D.	0.21	0.28	0.32	0.46	0.50
Weights of fetuses (g) (Females)	mean	3.82	3.88	3.82	3.87	3.89
	S.D.	0.21	0.31	0.30	0.43	0.49
Dead fetuses	n	0	0	0	0	0
Live Births Index (%)		100.0	100.0	100.0	100.0	100.0
Runts Index (%)	mean	0.3	0.0	0.3	0.0	0.0
b. Resorptions
Number per dam	mean	0.9	1.3	1.3	1.5	1.9
	S.D.	0.9	1.4	1.3	1.2	1.8
Early Resorption	mean	0.9	1.1	1.3	1.4	1.5
	S.D.	0.9	1.1	1.3	1.3	1.6
Late Resorption	mean	0.0	0.2	0.0	0.1	0.4
	S.D.	0.2	0.7	0.2	0.4	0.8
Resorption Index (%)	mean	5.7	8.3	7.6	8.8	12.4
Pre-implantation Loss Index (%)	mean	13.5	18.5	7.2	5.6	14.6
c. Implantations	mean	16.0	15.3	16.4	16.9	15.3
	S.D.	2.2	2.3	4.3	2.3	3.7
Post-implantation Loss Index (%)	mean	5.7	8.3	7.6	8.8	12.4
d. Placentae
Mean Weights (g)	mean	0.53	0.53	0.54	0.55	0.58
	S.D.	0.08	0.05	0.06	0.06	0.07
2.    Fetal Examinations
Malformed Fetuses (skel. + visc.)		1	0	1	0	0
Malformation Index (%) (skel. + visc.)		0.3	0.0	0.3	0.0	0.0
Malformed Fetuses (external)		1	0	0	0	0
Malformation Index (%) (skel. + visc.+external)		0.3	0.0	0.3	0.0	0.0
Litters with Malformations		1	0	1	0	0
Variations	Skel.	139	101	114	103	91
Variation Index, skel.	(%)	75.1	60.8	66.3	62.4	61.9
Anomalies	Skel.	34	46	35	40	34
Anomalies Index Skel.	(%)	18.4	27.7	20.3	24.2	23.1

Applicant's summary and conclusion

Conclusions:

A NOAEL of 75 mg/kg bw/day can be concluded when administering NP by oral gavage to female rats from day 6 to day 15 of gestation.
At 150 mg/kg bw/day 3 of 21 females showed affected kidneys or spleens. However, the dose level of 300 mg/kg caused clear maternal toxic effects like increased mortality, reduced body weight gain and food consumption. With regard to the embryo-fetal development the NOAEL is at or above 300 mg/kg bw/day.

Executive summary:

In a teratogenicity study according OECD 414, Nonylphenol was administered to Wistar Rats (112 pregnant rats and 2219 fetuses) by gavage at dose levels of 0, 75, 150, 300 mg/kg bw/day from day 6-15 of gestation.

Treatment of the pregnant females at a dose level of 75 mg/kg was without any general toxicological effect. At a dose level of 150 mg/kg only 3 of 21 females showed affected kidneys or spleens. A dose level of 300 mg/kg caused clear maternal toxic effects based on increased mortality, reduced body weight gain and food consumption.

With regard to the embryo-fetal development a NOAEL of >= 300 mg/kg was found. 

This teratogenicity study in the Wistar rats is acceptable and satisfies the guideline requirement for a teratogenicity study according OECD 414 in rats conducted under GLP.