7-[2-(2-hydroxymethylethoxy)methylethoxy]tetramethyl-3,6,8,11-tetraoxa-7-phosphatridecane-1,13-diol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

7-[2-(2-hydroxymethylethoxy)methylethoxy]tetramethyl-3,6,8,11-tetraoxa-7-phosphatridecane-1,13-diol

EC Number:

253-211-7

EC Name:

7-[2-(2-hydroxymethylethoxy)methylethoxy]tetramethyl-3,6,8,11-tetraoxa-7-phosphatridecane-1,13-diol

Cas Number:

36788-39-3

Molecular formula:

C18H39O9P

IUPAC Name:

7-[2-(2-hydroxymethylethoxy)methylethoxy]tetramethyl-3,6,8,11-tetraoxa-7-phosphatridecane-1,13-diol

Test material form:

other: liquid

Details on test material:

Tradename: Weston 430Batch #: MW3J19A389

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Appearance: clear, colorless liquid
- Source and lot/batch No.of test material: MOMENTIVE Niax Color Stabilizer CS-22 LF Lot
Number 15ASIB001
- Expiration date of the lot/batch: 14-Jan-2017
- Purity: 95.7%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: Stable for at least 18 days
FORM AS APPLIED IN THE TEST (if different from that of starting material)
- As a solution in Polyethylene glycol 400

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Age at study initiation: (P) approximately 12 weeks
- Weight at study initiation: (P) Males: 289-325 g Females: 180-217 g
- Fasting period before study: no
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): free access to Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK)
Limited, Oxon, UK.
- Water (e.g. ad libitum): free access to mains drinking water was supplied from polycarbonate bottles atta
ched to the cage.
- Acclimation period: 7 days during which time their health status was assessed.
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 ºC
- Humidity: 50 ± 20 %
- Air changes: at least 15 per hour
- Photoperiod: 12 hours continuous light and 12 hours dark via low intensity fluorescent lighting

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dosage Volume: 4 ml/kg
VEHICLE
- Concentration in vehicle: 0, 25, 75, 250 mg/ml in polyethylene glycol 400

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Representative samples of test item formulation were taken and analyzed for concentration of CS22- LF. The results indicate that the prepared formulations were within ± 92 to 109% of the nominal concentration confirming the accuracy and suitability of the formulation procedure.

Duration of treatment / exposure:

approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

0, 100, 300 or 1000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were chosen based on the results of previous toxicity work (including a Rat Oral Gavage Fourteen Day Range-Finding Toxicity Study)

Examinations

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes. Intergroup differences did not indicate any need for more formal gravimetric measurements.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five males and females selected from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, 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: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute 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 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, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

Hematology
The following parameters were measured: Hemoglobin, Erythrocyte count, Hematocrit, Erythrocyte indices (mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration), Total leukocyte count, Differential leukocyte count, Platelet count, Reticulocyte count, Prothrombin time, and Activated partial thromboplastin time.

Blood Chemistry
The following parameters were measured: Urea, Inorganic phosphorus, Glucose, Aspartate aminotransferase, Total protein, Alanine aminotransferase, Albumin Alkaline phosphatase, Albumin/Globulin ratio (by calculation), Creatinine, Sodium, Total cholesterol, Potassium, Total bilirubin, Chloride, Bile acids, Calcium.

Sacrifice and pathology:

Necropsy
Adult males were terminated on Day 43 or 44. Adult females and surviving offspring were terminated on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were terminated on or after Day 25 post coitum. Where possible for all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted. All adult animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Adrenals, Brain, Spleen, Heart, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid). The following organs were weighed from all remaining animals: Pituitary (post-fixation), Prostate and Seminal Vesicles, Epididymides, Testes, Ovaries, Uterus (weighed with Cervix).

Histopathology
Samples of the following tissues were removed from five males and five females selected from each dose group and preserved in buffered 10% formalin, except where noted: Adrenals, Muscle (skeletal), Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Rectum, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Skin (hind limb), Esophagus Spinal cord (cervical, mid thoracic, and lumbar), Eyes *, Spleen, Heart, Stomach, Ileum (including peyer’s patches), Jejunum, Thyroid/Parathyroid, Kidneys, Trachea, Liver, Thymus, Lungs (with bronchi)#, Urinary bladder, Lymph nodes (mandibular and mesenteric). The following tissues were preserved from all remaining animals: Ovaries, Pituitary, Prostate, Coagulating gland, Seminal vesicles, Epididymides ¿, Gross lesions, Testes ¿, Uterus & Cervix, Mammary gland, and Vagina.
* Eyes fixed in Davidson’s fluid
¿ preserved in Modified Davidsons fluid
# lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative.

All tissues from the five selected control and five 1000 mg/kg bw/day dose group animals and the single animal that died during the study were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues from the remaining control and 1000 mg/kg bw/day animals and any animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and all 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined. Due to effects observed at necropsy, the testes and epididymides from all males at 100 and 300 mg/kg bw/day were also processed. Following the results of histopathological assessments, livers were also examined from five males and five females at 100 and 300 mg/kg bw/day.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Statistics:

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.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows: Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed 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 nonhomogeneity of means, the data were analyzed 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 (non-parametric).

Data not analyzed by the Provantis data capture system were assessed separately using the SPSS
statistical package.

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, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day sporadic incidences of increased post-dosing salivation were observed for both sexes from Day 13. Similar increased post-dosing salivation was apparent for the majority of males and females receiving 300 mg/kg bw/day during Days 34 to 36. Increased post-doing salivation incidence observed in this study most probably represents occasional difficulties in dosing animals, differences in individual dosing techniques and slight differences in subjective calling levels for this observation and was considered to be of no toxicological significance.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Assessment of hematology parameters for females at 1000 mg/kg bw/day has to be treated with caution as the female animals at this dosage were in a different physiological state in comparison to the other
females on the study.

For females at 1000 mg/kg bw/day, haemoglobin, erythrocyte count and hematocrit were statistically significantly higher and platelets and neutrophils counts were statistically significantly lower than control. For erythrocyte and platelet counts, the majority of individual values were outside the historical control range.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For males at 1000 mg/kg bw/day, higher total cholesterol for males attained statistical significance compared to control. Although only two individual values for these treated animals were outside the historical control range, this finding may reflect slightly altered liver metabolism as indicated by increased liver weight and histopathological findings.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, overall motor activity was statistically significantly lower than control for both sexes.

There were no treatment-related changes in the behavioural parameters for either sex at 100, 300 or 1000 mg/kg bw/day.

No inter-group differences in sensory reactivity assessments were apparent for either sex.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

For males at 300 and 1000 mg/kg bw/day, absolute and body weight-relative liver weights were statistically significantly higher than control.

For males at 300 and 1000 mg/kg bw/day, absolute and body weight-relative testes and epididymal weights were statistically significantly lower than control.

For males at 300 and 1000 mg/kg bw/day, absolute and body weight-relative kidney weights for males were statistically significantly higher than control, with differences for body weight-relative values showing a dosage relationship.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, small and flaccid testes were observed for all males, with the majority also showing small epididymides. At 300 mg/kg bw/day, flaccid testes were observed for two males.

Enlargement of the liver was apparent for three males at 300 mg/kg bw/day and the majority of males at 1000 mg/kg bw/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Marked tubular degeneration within the testes was present in all males at 1000 mg/kg bw/day and, to a lesser extent, all males at 300 mg/kg bw/day. At 100 mg/kg bw/day, minimal tubular degeneration was present in the majority of males.

Aspermia in the epididymis was present in all males at 1000 mg/kg bw/day. Mild or moderate cellular debris was present in all males at 300 mg/kg bw/day and minimal cellular debris was present in some (5/12) males at 100 mg/kg bw/day.

At 1000 mg/kg bw/day, there was an increase in rarefaction in the cytoplasm of the liver in some (6/11) males.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Key result

Dose descriptor:

LOAEL

Remarks:

Systemic

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

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

Sex:

female

Basis for effect level:

behaviour (functional findings)

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Applicant's summary and conclusion

Conclusions:

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400) over the same treatment period and at the same dosage volume.

At 1000 mg/kg bw/day there was a clear effect on fertility leading to only a single pregnancy which appeared to be attributable to treatment-related effects on the testes and epididymides. Although pregnancy rate was satisfactory at lower dosages, treatment-related effects were apparent for the testes and epididymides at 300 mg/kg bw/day and the testes at 100 mg/kg bw/day. A No Observed Adverse Effect Level (NOAEL) for systemic toxicity and for reproduction of the male could not therefore be established.

For females, there were no findings apparent at 300 mg/kg bw/day that were considered to preclude this dosage from being a NOAEL for systemic toxicity or for reproduction.