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Toxicological information

Repeated dose toxicity: oral

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Administrative data

short-term repeated dose toxicity: oral
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 June 2012 - 06 August 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
according to guideline
other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
GLP compliance:
yes (incl. QA statement)
WIL Research Europe B.V., Hambakenwetering 7, 5231 DD ‘s-Hertogenbosch, The Netherlands

Test material

Constituent 1
Chemical structure
Reference substance name:
(4-nonylphenoxy)acetic acid
EC Number:
EC Name:
(4-nonylphenoxy)acetic acid
Cas Number:
Molecular formula:
2-(4-nonylphenoxy)acetic acid
Details on test material:
- Name of test material (as cited in study report): (4-nonylphenoxy) acetic acid
- Physical state: Yellow to brown viscous liquid
- Analytical purity: 97.4% (GC fingerprint)
- Expiration date of the lot/batch: 22 June 2014
- Storage condition of test material: At room temperature in the dark

Test animals

other: Crl:WI(Han) (outbred, SPF-Quality)
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: approx. 313 g (m) and 204 g (f)
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment

- Temperature (°C): 18 to 24
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
other: A 1:1 ratio of Polyethylene glycol 400 (specific gravity 1.125; Merck, Darmstadt, Germany) and water (Elix, Millipore S.A.S., Molsheim, France).
Details on oral exposure:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity or the density of the test substance.

- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe.
- Amount of vehicle (if gavage): 5ml/kg
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (11 June 2012), according to a validated method (Project 499642, BASF Project 05Y0836/11X473). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable when the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated when the coefficient of variation was ≤ 10%. Formulations were considered stable when the relative difference
before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 43-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 42, 48 (Group 1), 52 (Group 2) and 63 (Group 3) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Doses / concentrations
Doses / Concentrations:
20, 60, 200 mg/kg body weight
actual ingested
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of the 14-day dose range finding study (Project 499639; BASF Project 01R0836/11X416).


Observations and examinations performed and frequency:
At dose level allocation, 5 animals/sex/group were randomly selected for functional observations, locomotor activity, clinical pathology, organ weights (full list) and histopathology.

- Time schedule: twice daily

- Time schedule: Daily, detailed clinical observations were conducted for all animals after dosing at no specific time point, but within a similar time period after dosing for the respective animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed
outside the home cage in a standard arena.

- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of
mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

- Time schedule for collection of blood: on the day of scheduled post mortem examination between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight (with a maximum of 24 hours)
- How many animals: the selected 5 animals/sex/group
- Parameters checked: White blood cells, Differential leucocyte count, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration MCHC, Platelets, Prothrombin time, Activated Partial thromboplastin time

- Time schedule for collection of blood: on the day of scheduled post mortem examination between 7.00 and 10.30 a.m.
- Animals fasted: Yes
- How many animals: the selected 5 animals/sex/group
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids

- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period after clinical signs observations (incl. arena observations, if applicable) and before blood sampling.
- Dose groups that were examined: the selected 5 animals/sex/group.
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength, locomotor activity

OTHER: General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined (see section 7.8.1 and 7.8.2 for details).
Sacrifice and pathology:
All males and females were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated. Necropsy was conducted on the following days:
- Females which delivered: on Lactation Days 6-7.
- Females which failed to deliver (nos. 60, 66): Post-coitum Days 27-28 (females with evidence of mating)
- Males: Following completion of the mating period (a minimum of 28 days of dose administration).
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
- selected 5 animals/sex/group: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid.
- All remaining males: Epididymides, Testes

Samples of the following tissues and organs from all animals were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches [jejunum, ileum] if detectable, Brain - cerebellum, mid-brain, cortex, Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Coagulation gland, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skeletal muscle, Eyes (with optic nerve (if detectable) and Harderian gland), (Skin), Spinal cord -cervical, midthoracic, lumbar, (Male and Female mammary gland area), Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach (forestomach and glandular stomach), Ileum, Testes, Jejunum, Thymus, Kidneys, Thyroid including parathyroid if detectable, (Lacrimal gland, exorbital), (Tongue), (Larynx), Trachea, Liver, Urinary bladder, Lung, infused with formalin, Uterus, Lymph nodes - mandibular, mesenteric, Vagina, (Nasopharynx), All gross lesions, (Esophagus)

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

Of all males of the control and high dose group and all males suspected to be infertile additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The stomach of all selected animals of Groups 2 and 3 and the thyroids of all selected females of Group 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of all animals of Groups 1 and 4 and from all males that failed to sire and all females that failed to deliver healthy pups (20 mg/kg bw/day: nos. 20 and 60; 60 mg/kg bw/day: nos. 26 and 66). Special emphasis was placed on the stages of spermatogenesis and histopathology of interstitial cell structure.
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Details on results:
No mortality occurred during the study period. No clinical signs of toxicity were noted during the observation period.
At 200 mg/kg bw/day rales and piloerection were noted for two animals on a single day, and hunched posture and tremor were also noted for individual animals on single days. At the limited incidence observed, these were not considered to be toxicologically relevant. Incidental findings that were noted included chromodacryorrhea of the right eye, dull right eye and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Body weight gains were slightly decreased in males and females during the pre-mating and/or mating period.
Males at 200 mg/kg bw/day had slight but significantly lower body weight gain on Day 8 of the mating period, while females lost a small amount of weight on Day 8 of the premating period and had slightly lower weight gain on Day 1 of the mating period.

No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.
Relative food consumption was slightly lower for females at 200 mg/kg bw/day during the pre-mating period and over Days 4-7 and 14-20 on the post coitum period (only significantly different on Days 14-17). However, the difference from controls was only slight and this was not considered to be toxicologically relevant.
At 20 mg/kg bw/day relative food consumption was significantly higher than controls on lactation Days 1-4. The difference was attributable to high means for female nos. 52 and 59, and was not treatment related or toxicologically relevant.

No toxicologically relevant changes occurred in haematological parameters of treated rats.
Red blood cells and haematocrit values were significantly lower for males at 60 and 200 mg/kg bw/day and reticulocytes and red blood cell distribution width (RDW) were significantly higher for females at 200 mg/kg bw/day. However, in the absence of any relevant effects on any other related parameter, these were not considered to be toxicologically relevant.

Total thyroxine (T4) concentrations were significantly higher for animals of both sexes at 200 mg/kg bw/day, and cholesterol was significantly higher for females at this dose level. The statistically significant changes seen for males at 20 (increased aspartate aminotransferase [ASAT] and lower inorganic phosphate) and 60 mg/kg bw/day (lower inorganic phosphate) occurred in the absence of a treatment related distribution and were not considered to be toxicologically relevant.

Hearing ability, pupillary reflex (except for the dull eye in one rat), static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not have a relationship to treatment. Females at 200 mg/kg bw/day had lower total movements and ambulatory counts compared to controls (not statistically significant). This was mostly attributable to lower activity for these females over blocks 4-7. In the absence of any relevant clinical signs like lethargy, this was not considered to be toxicologically relevant.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

Males at 200 mg/kg bw/day had significantly higher absolute and relative liver, thyroid and kidney weights (relative liver weights were higher for females as well). Absolute and relative thyroid weights were higher for females at 60 and 200 mg/kg bw/day (not significantly higher at 200 mg/kg bw/day). High dose females also had significantly increased relative uterine weights compared to controls.
Absolute spleen and kidney weights were statistically significantly higher in males at 60 mg/kg bw/day. Since there was no dose-dependency or the difference to control values was minor, these findings were not considered to be toxicologically relevant. All other organ weights and organ to body weight ratios were similar to control levels.

Several reddish or dark red foci on the stomach glandular mucosa were noted for four males and irregular surface of the forestomach was noted for one male and one female at 200 mg/kg bw/day. These were likely related to the acidic nature of the test substance. At 60 mg/kg bw/day male no. 26 had reduced size of both testes and epididymides. This male’s infertility was confirmed at the microscopic examination. This was not considered to be treatment related. Macroscopic findings noted for control and treated animals included a greenish soft nodule on the left epididymis, pelvic dilation of the kidneys, enlarged mandibular lymph nodes, yellowish hard nodule on the epididymal adipose tissue, alopecia, several tan foci on the left clitoral gland, ectopic splenic tissue and an enlarged spleen. These remained within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related trend. These findings were therefore not considered to be toxicologically relevant.

Treatment-related microscopic findings were present in the stomach (both sexes) and thyroid gland (200 mg/kg bw/day, females)
- Hyperplasia of the squamous epithelium was recorded at 60 mg/kg bw/day in 1/5 males and 2/5 females (minimal) and at 200 mg/kg bw/day in 4/9 males (1 minimal, 2 slight and 1 moderate) and in 5/5 females (1 minimal, 3 slight and 1 moderate).
- Hemorrhage(s) of the glandular stomach (minimal-slight) was recorded in 4/9 males at 200 mg/kg bw/day.
Thyroid gland (females):
- Diffuse follicular hyperplasia/hypertrophy was recorded in 2/5 females of Group 1 (minimal), 1/5 of Group 2 and 3 (minimal) and 5/5 females of Group 4 (3 minimal and 2 slight).
In the stomach a minimal degree of hyperplasia of the squamous epithelium was considered to be within the normal background level and therefore not considered treatment-related. All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain.

Effect levels

Dose descriptor:
Effect level:
60 mg/kg bw (total dose)
Based on:
test mat.
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Based on the results presented, a No Observed Adverse Effect Level (NOAEL) of 60 mg/kg bw/day was derived.
Executive summary:

A GLP compliant combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in male and female Wistar Han rats at dose levels of 20, 50 and 200 mg/kg bw/day. Animals of the control group received the vehicle, a 1:1 ratio of Polyethylene glycol 400 and water, alone. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 43-56 days). The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (Males: Week 4; females: end of lactation), body weight and food consumption (at least at weekly intervals), clinical pathology (Males: Week 4; females: end of lactation), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Daily administration of test material at the dose of 200 mg/kg bw caused slightly reduced body weight gains in males and females. Liver and thyroid gland weights were increased in both sexes. Kidney weights were slightly increased in males. Macroscopically, foci and irregular surface of the stomach were observed. Histopathological examinations revealed slight to moderate hyperplasia of the squamous epithelium of the stomach in males and females and minimal to slight hemorrhages of the glandular stomach in males. In the thyroid of females minimal to slight diffuse follicular hypertrophy was observed. Examination of thyroid hormones revealed increased T4 levels in both sexes. No toxicologically relevant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, and clinical laboratory investigations). No compound-related adverse effects were observed at 60 mg/kg bw/day. In conclusion, treatment with (4-nonylphenoxy) acetic acid by oral gavage in male and female Wistar Han rats at dose levels of 20, 60 and 200 mg/kg bw/day revealed parental toxicity at 200 mg/kg body weight/day. Based on these results, the No Observed Adverse Effect Levels (NOAEL) was determined at 60 mg/kg bw/day.