Phenol, 4-nonyl-, branched

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented study equivalent to OECD 407 guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

minor deviations which did not affect the integrity or outcome of the study

GLP compliance:

yes

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

Constituent 2

Reference substance name:

Phenol, 4-nonyl-, branched

EC Number:

284-325-5

EC Name:

Phenol, 4-nonyl-, branched

Cas Number:

84852-15-3

Molecular formula:

C15H24O

IUPAC Name:

4-(2,4-dimethylheptan-3-yl)phenol

Details on test material:

- Name of test material (as cited in study report): Nonylphenol
- Substance type: pure active substance
- Physical state: clear, colourless, viscous liquid
- Substance type: pure active substance
- Physical state: no data
- Storage condition of test material: at ambient temperature in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate
- Strain: CRL:CD(SD) BR
- Age at study initiation: approx. 28 days
- Weight at study initiation: 164.7-200.9 g (males) and 140.7-162.5 g (females)
- Fasting period before study: no data
- Housing: in groups of five in stainless steel wire mesh cages suspended over cardboard-lined trays
- Diet: SQC Rat and Mouse Maintenance Diet No. 1, Expanded, Ground Fine ad libitum from Special Diets Services Ltd)
- Water: mains drinking water ad libitum
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per h): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: July 7, 1988 To: August 19, 1988

Administration / exposure

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Test article concentrations: The concentration of the test article in the diet was adjusted on the basis of the predicted mid-week group mean body weight and an estimate of the food consumption.
- Storage temperature of food: half of food stored at ambient temperature and half at -20 °C for use mid-week

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Verification of stability by analysis of samples of diet with concentrations of 555 and 13500 mg/kg. Sampling on the day of mixing and 4, 7 and 14 days after mixing of the diets stored under ambient conditions. Repeated stability analysis on diet containing 210 mg/kg test substance with sampling on the day of mixing and 2, 3, 6, 7, 8 and 14 days after mixing. There was a reduction in test article concentration after 4 days of storage. The nominal concentration was reduced by up to 18%. The reduction continued over the 15 days storage period. Howerver, the losses were considered acceptable provided exposure to room tmperature was minimised, therefore weekly diet preparation was split into two halves with one stored at -20 °C until use in the second half of the week.
- For analysis of homogeneity samples (from diets with test article concentrations of 210, 555 and 13500 mg/kg) were taken from three distinct areas of bulk mix
- Analysis of achieved concentrations were performed on day 1 of the study from all diets and showed to be accurately prepared (85 to 95% of nominal)

Duration of treatment / exposure:

28 days

Frequency of treatment:

continuously with the diet

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
25 mg/kg/d
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100 mg/kg/d
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
400 mg/kg/d
Basis:
nominal in diet

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly; in addition all animals were examined once daily for signs of ill health or overt toxicity
BODY WEIGHT: Yes
- Time schedule for examinations: before treatment on the first day of the study and at weekly intervals thereafter and at necropsy


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each cage of animals determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 4
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: Yes (about 18 h)
- How many animals: all animals
- Parameters: haemoglobin, mean cell volume, red blood cell count indices (packed cell volume, mean cell haemoglobin, mean cell haemoglobin concentration), total and differential white blood cell count, platelet count, reticulocyte count


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 4
- Animals fasted: Yes (halothane)
- How many animals: all
- Parameters: glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, alkaline phosphatase, plasma cholinesterase, sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea and total bilirubin


URINALYSIS: Yes
- Time schedule for collection of urine: in week 4
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes (about 15 h)
- Parameters: volume, pH, specific gravity, glucose, protein, total bilirubin, ketones, urobilinogen, blood, microscopy of spun deposits and reducung substances

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, full internal and external examinations
ORGAN WEIGHTS: determination of the organ weights of adrenals, kidneys, liver and testes with epididymides (paired organs were weighted seperately)
HISTOPATHOLOGY: Yes
Fixation of samples of the following tissues: adrenals, aorta, brain, caecum, colon, duodenum, eyes, heart, ileum, jejunum, kidneys, larynx and pharynx, liver, lungs, lymph nodes oesophagus, ovaries, pancreas, pitiutary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles skeletal muscle, spleen, sternum, stomach, testes with epididymides, thymus, thyropids, tongue, trachea, urinary bladder, uterus, vagina and all gross lesions.
From control and high dose animals the following tissue was examined histopathologically: adrenals, kidneys, liver, heart, lungs, ovaries, spleen, testis with epididymides and all gross lesions. Gross lesions, liver and kidney from low and intermediate dose animals were also examined histopathologically.

Statistics:

Data were processed, where appropriate, to give group mean values and standard deviations.

Body weights and body weight gains: Analysis of variance. Test for equality of variances between groups by Levene's test. Transformations may be applied to achieve variance stability. Comparisons between each treatment group and control using DUNNETT'S test. Analysis for dose-response by regression.

Organ weights: analysis of covariance [excluding kidney weights of one animal (low dose female) due to extreme weights]. Test for equality of variances between groups by Levene's test. Transformations may be applied to achieve variance stability. Adjusted means for treated groups were compared against control using DUNNETT'S test. Where there is significant heterogenicity among the gradients (P< 0.01), the gradient for each treated group will be compared with that for control using the T-test; means were compared.

Haematology and clinical chemistry parameters (except total white blood cell count and total bilirubin): analysis of variance
Total white blood cell count and total bilirubin: analysis non-parametrically: KRUSKAL-WALILIS non-parametric ANOVA together with the protected WILCOXON RANK-SUM TEST for each treatment group against control and the TERPSTRA-JONCKHEERE TEST for dose-response.

The following parameters were log transformed prior to analysis: mean cell haemaglobin concentration neutrophils (total and %), GOT, GPT, alkaline phosphatase, urea, creatinine and AG ratio.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

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

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinical signs or mortalities.

BODY WEIGHT AND WEIGHT GAIN
No apparent effect of treatment on the body weights of the low and mid dose animals. In the high dose group animals gained significantly less weight than the control (p<0.001).This was apparent from week 1 and was most marked in the males. At week 4, group mean body weights were less than those of the controls by 26% and 13% for males and females, respectively. During week 4 there was a loss of body weight; this was probably exacerbated by food deprivation before laboratory investigations. (Table 1)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There was no apparent effect of treatment on the food consumption of the low and mid dose group animals although mid dose females ate slightly less than the controls (up to 13%) throughout the study.The food consumption of the high dose group animals was less than that of the control animals throughout the study. This was most marked in the males, which consumed 19 to 40% less than the control males; females consumed 6 to 27% less than the control. It is suggested that at this dose level the diet was unpalatable. (Table 2)
Calculated compound consumption was variable, in the mid and high dose groups particular, owing to variations in food consumption but as generally in good agreement with the nominal dose levels of 25, 100 and 400 mg/kg bw/day. (Table 3)

HAEMATOLOGY
There were no changes in any of the haematology parameters in the low and mid dose group considered to be related to treatment.
The majority of the high dose group males had a higher red blood cell count (RBC) and lower mean cell volume (MCV) and mean cell haemoglobin (MCH). The group means were about 6% greater (for RBC) or less (for MCV and MCH) than those of the control, achieving statistical significance only for MCV( p< 0.5) and MCH (p< 0.01). However the individual values were within normal ranges expected and were not considered to be of toxicological significance. There was no apparent effect in the high dose females or in any of the other parameters measured.

CLINICAL CHEMISTRY
There was large individual animal variation in most of the clinical chemistry parameters.
In males of the high dose group total cholesterol levels were slightly higher and the group mean glucose level was lower than that of the control. However, these differences did not achieve statistical significance. Group mean urea level was higher in high dose males and females by 24% and 10% respectively when compared to the controls achieving statistical significance in males (p< 0.5). There was no apparent effect in the low and mid dose groups.
The uric acid level was generally lower in the mid and high dose animals, but with the female values being within the range of the control values. Group mean values for mid and high dose males were less than those of the controls by 17 % and 39% respectively,however without statistical significance.
The albumin/globulin ratio in the high dose males was significantly lower (p< 0.5) than that of the control group, however there were no significant changes in albumin or total protein levels.
There was an appartent dose-related decrease in plasma cholinesterase levels in the female animals. Group mean values were less than those of the control by 15, 18 and 22% for the low, mid and high dose groups, respectively, but did not achieve statistical significance. However, the individual values were within the range of the control values and the decrease in group means may be due to two of the control animals having slightly higher values. Therefore this change is considered to be of no toxicological significance.

URINALYSIS
There were no apparent treatment-related changes in urine parameters.

ORGAN WEIGHTS
The absolute kidney and liver weights of the low and mid dose males were greater than those of the control animals. Absolute kidney, liver and testis weights of the high dose males were less than those of the control; this reflects the effect on body weight. There were no changes in the absolute organ weights of treated females.
There was an increase of relative liver weights in all dose groups of the male animals compared to control values by about 20%. However the increases did not reach statistical significance and are not in a clear dose-related manner. With regard to historical controls (2.8457 +/- 0.3634) the relative liver weights of the control group are very low but within the normal range . The liver weights of the low and mid dose are also within the normal range of historical control data. Therefore only the relative liver weights of the high dose group show a slightly test substance-related increase compared to controls.
There was a dose-related increase in group mean relative kidney weights of the male animals, in the high dose group about 22% greater than control values. The differences in kidney weights achieved statistical signifance in low and mid dose males (p< 0.05 and p< 0.1). However , compared to historical controls show the values being in the normal range. Merely the increases in the high dose group can be attributed to the test substance.
Relative adrenals weights of themales were increased in all treatment groups compared to controls by about 18%. But the increased did not show a clear dose relation and did not reach statistical significance. Compared to historical controls all values were within the typical normal range and therefore the increases can not be attributed to the test substance and are of no toxicological relevance.
High dose group testis weights were 23% greater than those of the controls.
There was no effect on the relative organ weights of treated females. (Table 3)


GROSS PATHOLOGY
No treatment related effects.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related effect in females. In the males there was a minor vacuolation in the periportal hepatocytes in the high dose group.
There was a dose-related hyaline droplet accumulation in the renal proximal tubules in the males. Review of the historical control data show that hyaline droplets occured in many histrocal controls of the same extent than in the low and mid dose group. Only the high dose group shows a substance-specific increase of this effect. The observed changes are propably a matter of a specific nephropathy of male rats which are not attributable to humans and are therefore of no relevance.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No treatment related effects.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: overall effects

2

Dose descriptor:

LOAEL

Effect level:

400 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: changes in body weight gain and food consumption in both sexes; and certain clinical chemistry parameters, changes in relative organ weights of kidney, liver and testes and a minor vacuolation in the periportal hepatocytes found in males only.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Group mean body weight (g, mean values and standard deviations)
Group		Control	25 mg/kg/d	100 mg/kg/d	400 mg/kg/d
	Sex
Start	male	189.6 ± 5.60	187.5 ± 3.74	190.8 ± 9.53	185.0 ± 13.16
Week 1	male	235.8 ± 3.42	234.6 ± 2.48	240.6 ± 10.86	215.0 ± 17.60
Week 2	male	288.3 ± 6.04	284.8 ± 6.51	291.0 ± 10.64	238.7 ± 15.21
Week 3	male	335.5 ± 9.15	328.1 ± 5.85	333.7 ± 9.21	268.0 ± 19.69
Week 4	male	343.8 ± 10.56	339.0 ± 6.26	341.5 ± 8.01	254.1 ± 26.58
Weight gain (g) start to week 4	male	66.3 ± 4.13	65.3 ± 7.30	62.4 ± 2.54	35.4 ± 6.30***
Start	female	150.3 ± 9.11	151.9 ± 6.07	151.2 ± 6.98	152.1 ± 5.06
Week 1	female	174.0 ± 10.60	173.1 ± 7.89	172.9 ± 12.48	162.7 ± 3.99
Week 2	female	194.3 ± 11.14	191.8 ± 9.27	192.5 ± 12.35	180.6 ± 4.87
Week 3	female	211.7 ± 12.07	217.2 ± 9.51	213.6 ± 8.12	187.5 ± 4.60
Week 4	female	216.6 ± 12.70	217.2 ± 9.51	213.6 ± 8.12	187.5 ± 4.60
Weight gain (g) start to week 4	female	154.2 ± 12.07	151.6 ± 2.94	150.7 ± 2.96	69.1 ± 24.10***
*** p< 0.0001
Table 2: Group mean food consumption (g/animal/week)
Group		Control	25 mg/kg/d	100 mg/kg/d	400 mg/kg/d
	Sex
Week 1	male	164.1	177.3	181.8	132.6
Week 2	male	201.0	206.2	207.3	156.7
Week 3	male	185.9	187.1	191.8	138.6
Week 4	male	183.7	176.3	175.4	110.3
Week 1	female	124.7	117.9	138.3	116.6
Week 2	female	143.4	124.9	152.6	133.0
Week 3	female	134.4	128.4	137.1	114.1
Week 4	female	138.4	125.9	145.8	100.6
Table 3: Group mean compound consumption (mg/kg/d)
Group		Control	25 mg/kg/d	100 mg/kg/d	400 mg/kg/d
	Sex
Week 1	male	0.0	28.0	112.4	353.6
Week 2	male	0.0	28.9	113.9	479.2
Week 3	male	0.0	22.9	93.6	350.0
Week 4	male	0.0	24.7	96.2	344.4
Week 1	female	0.0	23.2	109.2	379.2
Week 2	female	0.0	26.7	111.0	446.5
Week 3	female	0.0	25.7	90.0	348.6
Week 4	female	0.0	25.1	110.6	368.2
Table 4: Group mean organ/body weight ratios (%) of males (mean values and standard deviations)
Group		Control	25 mg/kg/d	100 mg/kg/d	400 mg/kg/d
Liver		2.6984 ± 0.1293	2.9191 ± 0.2735	2.8950 ± 0.1464	3.2562 ± 0.2815
Adrenal - left		0.0081 ± 0.0010	0.0089 ± 0.0022	0.0094 ± 0.0006	0.0095 ± 0.0011
Adrenal - right		0.0070 ± 0.0008	0.0081 ± 0.0014	0.0083 ± 0.0001	0.0083 ± 0.0009
Kidney - left		0.3165 ± 0.0098	0.3428 ± 0.0152	0.3550 ± 0.0199	0.3854 ± 0.0244
Kidney - right		0.3190 ± 0.0141	0.3593 ± 0.0203	0.3615 ± 0.0176	0.3895 ± 0.0280
testis with epididymides - left		0.6446 ± 0.0238	0.6780 ± 0.0392	0.6053 ± 0.1627	0.7946 ± 0.0564
testis with epididymides - right		0.6434 ± 0.0316	0.6796 ± 0.0316	0.6421 ± 0.1032	0.7927 ± 0.0644

Applicant's summary and conclusion

Conclusions:

The LOAEL in a 28 day study in rat is 400 mg/kg bw/day based on changes in body weight gain and food consumption in both sexes; and certain clinical chemistry parameters, changes in relative organ weights of kidney, liver and testes and a minor vacuolation in the periportal hepatocytes found in males only.

Executive summary:

In a subacute toxicity study Nonylphenol (>= 98%) was administered to 5 SD rats/sex/dose in diet at dose levels of 0, 25, 100, 400 mg/kg bw/day.

There were no mortalities or treatment related clinical signs of toxicity. At 400 mg/kg/day, body weight gain, food consumption and food utilization was significantly reduced throughout the study for both sexes. For males only at 400 mg/kg/day there were slight deviations in certain clinical chemistry parameters (urea and cholesterol levels were increased; glucose levels were reduced). There were increases in the group mean relative kidney, liver and testes weights (all by about 20% compared with controls). Histopathological examination revealed hyaline droplet accumulation in the renal proximal tubules (an effect considered to be of no relevance to human health) and a minor vacuolation in the periportal hepatocytes for males at 400 mg/kg/day. Among the females at this level, there were no treatment-related changes in the organs.

 

The LOAEL is 400 mg/kg bw/day, based on body weight gain and food consumption. The NOAEL is 100 mg/kg bw/day.

 

This subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a subacute oral study OECD 407 in rat.