Poly(oxy-1,2-ethanediyl), .alpha.-[3-[(carboxymethyl)methylamino]-2-hydroxypropyl]-.omega.-(4-nonylphenoxy)-, sodium salt (1:1)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

other information

Study period:

The study was performed between 12 October 2012 and 06 May 2013. The in-life phase of the study was conducted between 26 October 2012 (first day of treatment) and 23 November 2012 (final day of necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nonylphenol ethoxylate, sarcosine derivative

IUPAC Name:

Nonylphenol ethoxylate, sarcosine derivative

Constituent 2

Reference substance name:

disodium 2-({2-hydroxy-3-[2-(4-nonylphenoxy)ethoxy]propyl}(methyl)amino)acetate 2-{[3-({1-chloro-3-[2-(4-nonylphenoxy)ethoxy]propan-2-yl}oxy)-2-hydroxypropyl](methyl)amino}acetate

EC Number:

616-248-3

Cas Number:

75627-31-5

Molecular formula:

UVCB not applicable

IUPAC Name:

disodium 2-({2-hydroxy-3-[2-(4-nonylphenoxy)ethoxy]propyl}(methyl)amino)acetate 2-{[3-({1-chloro-3-[2-(4-nonylphenoxy)ethoxy]propan-2-yl}oxy)-2-hydroxypropyl](methyl)amino}acetate

Test material form:

other: Pale yellow liquid

Details on test material:

Sponsor's identification : Nonylphenol ethoxylate, sarcosine derivative CAS no. 75627-31-5
Description : Pale amber coloured liquid/ Pale yellow liquid
Purity : 81.7%
Batch number : 120229AAH
Label : NP SARCOSINAT_H Nonylphenol ethoxylate, sarcosine derivative (75627-31-5) Batch No 120229AAH
Date received : 06 June 2012 / 11 October 2012
Storage conditions : Ambient, in the dark
Expiry date : 28 February 2017

The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.
A Certificate of Analysis of the Test Item is included as Addendum 6. (attachment 1)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK.
- Age at study initiation: Approximately six to eight weeks old.
- Weight at study initiation: At the start of treatment the males weighed 211 to 248g, the females weighed 150 to 168g.
- Fasting period before study: No data.
- Housing: The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet: ad libitum, pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK).
- Water: ad libitum, mains drinking water was supplied from polycarbonate bottles attached to the cage.
Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for eight days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility.
- Temperature (°C): 22 ± 3ºC, there were no deviations from these ranges.
- Humidity (%): 50 ± 20%, there were no deviations from these ranges.
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): Low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: The in-life phase of the study was conducted between 26 October 2012 (first day of treatment) and 23 November 2012 (final day of necropsy).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

, distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least twelve days. Formulations were therefore prepared weekly during the treatment period and stored at
approximately 4ºC in the dark.
Dosages were selected based on available toxicity data including a seven day rat rangefinding toxicity study.
In the range-finding study, there were no findings observed at 1000 mg/kg bw/day AI that were considered sufficient to exclude this dosage from use as
a high dosage in this main twenty-eight day toxicity study. Lower dosages were selected taking into consideration the Globally Harmonised System of Classification and Labelling of Chemicals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water was successfully used on the range-finding study and the same vehiclele, at a dosage volume of 5 ml/kg body weight, was therefore employed in this main study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken and analysed for concentration of Nonylphenol ethoxylate, sarcosine derivative CAS no. 75627-31-5 at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 16 (Attachment 2). The results indicate that the prepared formulations were within ± 8% of the nominal concentration.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
30 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Male and Female: 5 animals per sex at 0 mg/kg/day
Male and Female: 5 animals per sex at 30 mg/kg/day
Male and Female: 5 animals per sex at 300 mg/kg/day
Male and Female: 5 animals per sex at 1000 mg/kg/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.
The cage distribution within the holding rack was also randomised.

The volume of test and control item administered to each animal was based on the most recent body weight and was adjusted at weekly intervals.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. All observations were recorded.

BODY WEIGHT: Yes
Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also performed prior to terminal kill.

FOOD CONSUMPTION:
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

FOOD EFFICIENCY:
Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION: Yes
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Week 3 where water intake was measured gravimetrically. As a possible intergroup difference was detected during Week 3, water consumption was continued to be measured and recorded for each cage group until the termination of the study.

OPHTHALMOSCOPIC EXAMINATION: No

Laboratory Investigations
Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

HAEMATOLOGY: Yes
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
.................................. - mean corpuscular volume (MCV)
.................................. - mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
................................................. - lymphocytes (Lymph)
................................................. - monocytes (Mono)
................................................. - eosinophils (Eos)
................................................. - basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

CLINICAL CHEMISTRY: Yes
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea, Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 27, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.
This test was developed from the methods used by Irwin (1968) and Moser et al (1988).

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The following parameters were observed:
Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

Sacrifice and pathology:

On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

GROSS PATHOLOGY: Yes
Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Liver, Brain, Ovaries, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Pituitary (post-fixation), Thyroid/Parathyroid, Prostate and Seminal Vesicles (with coagulating glands and fluids), Uterus with Cervix.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin except where stated:
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Rectum, Salivary glands (submaxillary), Caecum, Sciatic nerve, Colon, Seminal vesicles (with coagulating glands and fluids), Duodenum, Epididymides ♦, Skin (hind limb), Eyes *, Spinal cord (cervical, mid-thoracic and lumbar), Gross lesions, Heart, Spleen, Ileum (including peyer’s patches), Stomach, Jejunum, Testes ♦, Kidneys, Thymus, Liver, Thyroid/Parathyroid, Lungs (with bronchi)#, Trachea, Lymph nodes (mandibular and mesenteric), Urinary bladder, Mammary gland, Uterus & Cervix, Muscle (skeletal), Vagina, Oesophagus.
♦ = preserved in Bouin’s fluid then transferred to Industrial Methylated Spirits (IMS) approximately 48 hours later
* = eyes fixed in Davidson’s fluid
# = Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Apart from the Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Salivary glands (submaxillary), Skin (hind limb) and Oesophagus, tissues from all control and 1000 mg/kg bw/day AI dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Haematoxylin and Eosin for subsequent microscopic examination.

All tissues were despatched to the histology processing Test Site (Harlan Laboratories Ltd., Zelgliweg 1, CH-4452 Itingen, Switzerland) for processing. Any macroscopically observed lesions were also processed together with the liver and spleen from all 30 and 300 mg/kg bw/day AI dose group animals. In addition, sections of testes and epididymides from all Control and 1000 mg/kg bw/day AI males were stained with Periodic Acid-Schiff (PAS) stain and examined.
Since there were indications of treatment-related thyroid changes, examination was subsequently extended to include similarly prepared sections of thyroid from all animals from the low and intermediate groups.

Other examinations:

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -20°C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were not processed and will be discarded on issue of the final report.

Statistics:

Data were processed to give summary incidence or group mean and standard deviation values were appropriate. All data were summarised in tabular form.
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no unscheduled deaths. Please see "details on results" below for further information.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no unscheduled deaths. Please see "details on results" below for further information.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects of treatment on body weight or body weight gain for either sex at 30, 300 or 1000 mg/kg bw/day AI. Statistical analysis of the data did not reveal any significant intergroup differences.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Please see "details on results" below for further information.

Food efficiency:

no effects observed

Description (incidence and severity):

Please see "details on results" below for further information.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Please see "details on results" below for further information.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Please see "details on results" below for further information.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Please see "details on results" below for further information.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Please see "details on results" below for further information.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Please see "details on results" below for further information.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Please see "details on results" below for further information.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Please see "details on results" below for further information.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Information on Tables can be found in Attachment 3 - Tables 1- 14.
Information on Figures can be found in Attachment 4 - Figures 1 - 4.

CLINICAL SIGNS AND MORTALITY
A summary incidence of daily clinical observations is given in Table 1.
Post-dosing salivation was observed for both sexes at 300 and 1000 mg/kg bw/day AI. At both dosages, increased salivation was evident in all animals from the first week of treatment.
Additionally one female at 300 mg/kg bw/day AI and one male and two females at 1000 mg/kg bw/day AI also showed noisy respiration on an occasion during the study.
One female at 1000 mg/kg bw/day AI also showed gasping respiration immediately after dosing on Day 15 of the study but appeared fully recovered one hour later.
There were no clinical signs apparent for either sex at 30 mg/kg bw/day AI or for control animals.

BODY WEIGHT AND WEIGHT GAIN
Group mean weekly body weights and standard deviations are given in Table 6 and are presented graphically in Figure 1 and Figure 2. Group mean weekly body weight gains and standard deviations are given in Table 7.

FOOD CONSUMPTION
Group mean weekly food consumptions are given in Table 8 and are presented graphically in Figure 3 and Figure 4. Weekly food efficiencies are given in Table 9.
There was no obvious effect of treatment on food consumption for either sex at 30, 300 or 1000 mg/kg bw/day AI.

FOOD EFFICIENCY
Weekly food efficiencies are given in Table 9.
There was no obvious effect of treatment on food conversion efficiency for either sex at 30, 300 or 1000 mg/kg bw/day AI.
At 1000 mg/kg bw/day AI food conversion for both sexes was slightly lower than control during the final week of treatment. This was consistent with a slightly lower body weight gain observed during this period. Previous food efficiency was similar or slightly superior to controls and therefore the intergroup difference was considered to be incidental and of no toxicological significance.

WATER CONSUMPTION
Group mean daily water consumptions are given in Table 10.
At 1000 mg/kg bw/day AI, water consumption for females was higher than control throughout the last two weeks of the study (the period of formal measurement of water intake).
For males at 1000 mg/kg bw/day AI and both sexes at 30 and 300 mg/kg bw/day AI there were no consistent differences in water consumption compared to control that indicated any clear effect of treatment.

HAEMATOLOGY
Group mean values and standard deviations for test and control group animals are given in Table 11 (statistically significant differences are indicated).
For females at 300 and 1000 mg/kg bw/day AI, total leucocyte count was statistically significantly higher than controls, primarily due to a statistically significant increase in lymphocyte count. The majority of the individual values for these parameters were outside of the historical control ranges for rats of the strain and aged used.
No toxicologically significant effects were detected in males at 1000 or 300 mg/kg bw/day AI or for either sex at 30 mg/kg bw/day AI.
For males at 300 and 1000 mg/kg bw/day AI, mean cell haemoglobin concentration and activated partial thromboplastin times were lower than control with differences attaining statistical significance. Additionally for males at 1000 mg/kg bw/day AI, haemoglobin levels were also statistically significantly lower than control. The majority of individual values were within the normal ranges for rats of the strain and age used and in the absence of any histopathological correlates the intergroup differences were considered not to be of toxicological significance.
For males at all dosages, total leucocyte count was lower than control principally due to a decrease in the numbers of lymphocytes; differences attained statistical significance but showed no dosage relationship. The majority of individual values were also within the normal ranges for rats of the strain and age used therefore the intergroup differences were considered not to be of toxicological importance.

CLINICAL CHEMISTRY
Group mean values and standard deviations for test and control group animals are given in Table 12 (statistically significant differences are indicated).
For females at 1000 mg/kg bw/day AI, total protein was statistically significantly lower than control. Although there were no statistically significant differences detected for albumin or albumin/globulin ratio the majority of individual values were outside of the historical control ranges for rats of the strain and aged used. Females at 300 and 1000 mg/kg bw/day AI, also showed statistically significantly higher cholesterol and bile acids than control.
No statistically significant differences from control were apparent for blood chemistry parameters for males at all dosages or in females at 30 mg/kg bw/day AI.

NEUROBEHAVIOUR
Functional Observations
A summary incidence of behavioural assessments is given in Table 2 and group mean behavioural assessment scores are given in Table 3. Group mean functional performance test values and standard deviations are given in Table 4 (statistically significant differences are indicated). Group mean sensory reactivity assessments are given in Table 5.

Behavioural Assessments
Weekly open field arena observations did not reveal any treatment related effects in treated animals when compared to controls.
All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used, and the intergroup differences were considered to be of no toxicological importance.

Functional Performance Tests
No toxicologically significant effects of treatment were detected in grip strength or motor activity assessments for treated animals when compared to controls.
Males treated with 1000 and 300 mg/kg bw/day AI showed a statistically significant reduction in overall activity. In the absence of a true dosage response or any clinical observations to suggest an effect on neurotoxicity, the intergroup differences were considered to be of no toxicological importance.

Sensory Reactivity Assessments
There were no toxicologically significant changes in sensory reactivity.
All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

ORGAN WEIGHTS
Group mean absolute and body weight-relative organ weights and standard deviations for test and control group animals are presented in Table 14 (statistically significant differences are indicated).
For males at 1000 mg/kg bw/day AI absolute and body weight relative liver weights were higher than control with differences attaining statistical significance.
There were no other effects of treatment on absolute or body weight relative organ weights for either sex at 30, 300 or 1000 mg/kg bw/day AI.
For females at 300 mg/kg bw/day AI mean absolute and body weight relative pituitary weights were statistically significantly higher than control. In the absence of any dosage relationship or histopathology correlates this finding was considered incidental and unrelated to treatment.

GROSS PATHOLOGY
A summary incidence of necropsy findings is given in Table 13.
No treatment related macroscopic abnormalities were detected.
Macroscopic necropsy findings were restricted to small and flaccid testes and small epididymides for one male receiving 1000 mg/kg bw/day AI. These findings correlated histopathologically with testicular bilateral germinal epithelial atrophy and were considered to be within the range of normal background alterations and therefore, considered not to be of toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver: In comparison to controls there was hypertrophy of centrilobular hepatocytes in 3/5 males given 300 mg/kg/day AI and all the males and 3/5 females given 1000 mg/kg/day AI. The finding was graded as mild or moderate in males given the highest dosage and was considered to correlate with the increase in mean absolute and relative liver weights seen in this group. The finding was graded as minimal in all the other affected animals. The morphological appearance was consistent with a diagnosis of hepatic enzyme induction that, in the absence of any other morphological evidence of liver toxicity, was considered to be an adaptive and non-adverse finding.
Thyroid Glands: In all the males given 300 mg/kg/day AI and all the males and 3/5 females given 1000 mg/kg/day AI there was minimal or mild diffuse hypertrophy of the follicular epithelium. The most likely explanation for the hypertrophy was considered to be increased hepatic clearance of thyroid hormone, due to hepatic enzyme induction, leading to increased circulating thyroid stimulating hormone (TSH). The single minimal occurrence of hypertrophy in a male given 30 mg/kg/day AI was probably a chance finding due to individual variation rather than a treatment-related effect at this dosage.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 other: mg/kg bw/day AI

Based on:

test mat.

Sex:

male/female

2

Dose descriptor:

NOEL

Effect level:

30 other: mg/kg bw/day AI

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; haematology; histopathology;

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

DISCUSSION

The oral administration of Nonylphenol ethoxylate, sarcosine derivative CAS no. 75627- 31-5 to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day AI, resulted in treatment related findings in animals of either sex treated with 1000 and 300 mg/kg bw/day AI.

Clinical observations were confined to increased salivation observed for both sexes at 300 and 1000 mg/kg bw/day AI. At both dosages all animals were affected but the overall incidence tended to be higher at 1000 mg/kg bw/day AI. Increased overall water consumption was also evident in females from these dosages. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to be related to systemic toxicity.

Haematological examinations revealed an increase in total leucocyte count and lymphocyte count for females at 1000 and 300 mg/kg bw/day AI. Although the majority of individual values were outside of the historical control range, in the absence of any evidence of histopathological change in the bone marrow, these isolated findings were considered not to represent an adverse effect of treatment.

Absolute and relative liver weights were elevated in males treated with 1000 mg/kg bw/day AI. Associated treatment related microscopic findings were evident in the liver. Microscopic examination of liver sections revealed centrilobular hepatocellular hypertrophy in animals of either sex treated with 1000 mg/kg bw/day AI and in males treated with 300 mg/kg bw/day AI. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of any associated inflammatory or degenerative changes, is generally considered to be adaptive in nature and does not represent an adverse health effect in rodents. Blood chemical examinations revealed a reduction in total protein in females treated with 1000 mg/kg bw/day AI and an increase in cholesterol and bile acid in females treated with 1000 and 300 mg/kg bw/day AI. Although some of the individual values were outside of the normal ranges for rats of the strain and age used these findings were most likely to be associated with the adaptive metabolic changes.

Microscopic examinations of the thyroid revealed diffuse hypertrophy of the follicular epithelium in animals of either sex treated with 1000 mg/kg bw/day AI and in males treated with 300 mg/kg bw/day AI. This finding is considered to be a secondary response to the hepatocellular hypertrophy. It is well documented that an increased metabolism by the liver of the thyroid hormones (thyroxine and triiodothyronine) causes an increased release of TRH (thyrotropin releasing hormone) from the hypothalamus and TSH (thyrotropin stimulating hormone) from the pituitary and consequent activation of the thyroid epithelium (Hypothalamic-Pituitary-Thyroid Axis). The thyroid of rats is highly sensitive to this phenomenon which is particularly species-specific with no safety relevance for humans and is generally regarded as adaptive in nature and not an adverse effect of treatment.

Based upon the results of this study whilst there is evidence of response to the administration of the test item, observations of post dose saturation plus the histopathological changes to liver and thyroid organs with attendant blood chemistry alterations, it should be noted that all these findings can be classified as adaptive responses. None of the findings are associated with degenerative organ change and none represent a significant alteration to the physiological status of the animal. None of the findings would be classified as an adverse effect upon the treated animal.

Overall it was considered that the No Observed Adverse Effect Level (NOAEL) for this study was 1000 mg/kg bw/day AI and the no observed effect level (NOEL) was 30 mg/kg bw/day AI.

Applicant's summary and conclusion

Conclusions:

The oral administration of Nonylphenol ethoxylate, sarcosine derivative CAS no. 75627- 31-5 to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day AI, resulted in treatment related findings in animals of either sex treated with 1000 and 300 mg/kg bw/day AI.
The 'No Observed Effect Level' (NOEL) for either sex was therefore considered to be 30 mg/kg bw/day AI.
The effects detected at 1000 and 300 mg/kg bw/day AI however were confined to increases in total leucocyte count and lymphocyte count (females only), reduced total protein (females only), increased bile acid and cholesterol (females only) and adaptive microscopic liver and thyroid changes. These were considered not to represent an adverse effect of treatment therefore the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 1000 mg/kg bw/day AI for either sex.

Executive summary:

Introduction. The study was designed to investigate the systemic toxicity of the test item. It is compatible with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

This study was also designed to be compatible with the requirements of Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods. The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 1000 mg/kg bw/day AI (incorporating a correction factor for 81.7% purity). A control group of five males and five females was dosed with vehicle alone (Distilled water) over the same treatment period.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Mortality. There were no unscheduled deaths on the study.

Clinical Observations. Post-dosing salivation was observed for both sexes at 300 and 1000 mg/kg bw/day AI; at both dosages all animals were affected but the overall incidence tended to be higher at 1000 mg/kg bw/day AI. Additionally one female at 300 mg/kg bw/day AI and one male and two females at 1000 mg/kg bw/day AI also showed noisy respiration on an occasion during the study.

One female at 1000 mg/kg bw/day AI also showed gasping respiration immediately after dosing on Day 15 only.

There were no clinical signs apparent for either sex at 30 mg/kg bw/day AI.

Behavioural Assessment. There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests. There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity.

Body Weight. Body weight gain for both sexes was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day AI.

Food Consumption. Food consumption and food conversion efficiency was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day AI.

Water Consumption. At 1000 and 300 mg/kg bw/day AI, water consumption for females was higher than control throughout the last two weeks of the study.

For males at 1000 and 300 mg/kg bw/day AI and both sexes at 30 mg/kg bw/day AI there were no consistent differences in water consumption compared to control.

Haematology. At 1000 and 300 mg/kg bw/day AI, females showed higher total leucocyte count and lymphocyte count than controls.

For males at 1000 and 300 mg/kg bw/day AI and both sexes at 30 mg/kg bw/day AI, no toxicologically significant effects were detected.

Blood Chemistry. At 1000 and 300 mg/kg bw/day AI, females showed higher cholesterol and bile acid values than controls. Females at 1000 mg/kg bw/day AI also showed a reduction in total protein.

For males at 1000 and 300 mg/kg bw/day AI and both sexes at 30 mg/kg bw/day AI, no treatment-related effects were detected. Necropsy. No treatment-related macroscopic abnormalities were detected.

Organ Weights. For males at 1000 mg/kg bw/day AI absolute and body weight relative liver weights were higher than control.

There were no other effects of treatment on absolute or body weight relative organ weights for either sex at 30, 300 or 1000 mg/kg bw/day AI.

Histopathology. The following treatment related microscopic findings were detected:

Liver: hypertrophy of centrilobular hepatocytes was evident in animals of either sex treated with 1000 mg/kg bw/day AI and in males treated with 300 mg/kg bw/day AI. The finding was graded as mild or moderate in males given the highest dosage and was graded as minimal in all the other affected animals.

Thyroid Glands: minimal or mild diffuse hypertrophy of the follicular epithelium was evident in animals of either sex treated with 1000 mg/kg bw/day AI and in males treated with 300 mg/kg bw/day AI.

Conclusion. The oral administration of Nonylphenol ethoxylate, sarcosine derivative CAS no. 75627-31-5 to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day AI, resulted in treatment related findings in animals of either sex treated with 1000 and 300 mg/kg bw/day AI.

The 'No Observed Effect Level' (NOEL) for either sex was therefore considered to be 30 mg/kg bw/day AI.

The effects detected at 1000 and 300 mg/kg bw/day AI however were confined to increases in total leucocyte count and lymphocyte count (females only), reduced total protein (females only), increased bile acid and cholesterol (females only) and adaptive microscopic liver and thyroid changes. These were considered not to represent an adverse effect of treatment therefore the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 1000 mg/kg bw/day AI for either sex.