Reaction mass of Phenol, dodecyl-, sulfurized, calcium salts and benzene and butene and calcium carbonate and calcium dihydroxide and sulphur trioxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A GLP study conducted in accordance with OECD test guideline.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Recovery and Endocrine Assessments were included in the study design.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phenol, paraalkylation products with C12-rich branched olefins derived from propene oligomerisation, reaction products with sulphur monochloride and decene, reaction products with polybutenyl benzenesulphonic acid, carbon dioxide and calcium hydroxide

EC Number:

903-161-3

IUPAC Name:

Phenol, paraalkylation products with C12-rich branched olefins derived from propene oligomerisation, reaction products with sulphur monochloride and decene, reaction products with polybutenyl benzenesulphonic acid, carbon dioxide and calcium hydroxide

Test material form:

liquid: viscous

Details on test material:

-Test Article I.D.- EC-903-161-3
- Physical state: Dark brown, opaque, viscous liquid
- Lot/batch No.: 1365549-31/03 08:24-206698
- Sub-samples :E00037-107, E00009-215
- Expiration date of the lot/batch: 21 May 2012 for both
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Supplier: Infineum UK Ltd., Steventon, Oxon, United Kingdom
*EC 903-161-3 is exclusively synthesised and handled in solvent oil. Therefore testing was conducted on sample that contained 40.2% base oil and 59.8 % EC 903-161-3.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

Each rat was uniquely identified by a Monel® metal ear tag displaying the animal number.

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 12 weeks old
- Weight at study initiation: (P) Males: 363 g to 478 g; Females: 221 g to 285 g
- Housing: Until pairing, all F0 animals were housed individually in clean, stainless steel wire mesh cages suspended above cage board. The 5 rats/sex assigned to the post-treatment phase were not paired and remained in clean, stainless steel wire mesh cages until euthanasia. The breeding phase rats (10/sex) were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding. Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until post-cohabitation or post-mating day 25.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except during the period of fasting prior to clinical pathology blood collection
- Water: Reverse osmosis purified (on site) drinking water, delivered by an automatic watering system ad libitum
- Acclimation period: 16 days prior to the first day of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 70.9 to 71.8 deg F (21.6 to 22.1 deg C)
- Humidity: 49.9 to 65.4%
- Air changes: 10 changes/hr
- Photoperiod: Fluorescent lighting provided illumination for a 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod

IN-LIFE DATES: From: 15 June 2010 To: 23 August 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: mineral oil, USP

Details on exposure:

The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible teflon®-shafted, ball-tipped dosing cannula (Natume, Japan) once daily.

PREPARATION OF DOSING SOLUTIONS: The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group. The vehicle was mixed throughout preparation, sampling, and dose administration procedures, and was warmed to 60°C for a minimum of 30 minutes prior to daily dispensation. The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures, and were warmed to 60°C for a minimum of 30 minutes prior to daily dispensation.

VEHICLE
The vehicle used in preparation of the test item formulations and for administration to the control group was mineral oil, USP.
- Concentration in vehicle: 0, 6, 20, or 200 mg/ml (corrected for % active ingredient)
- Dosage volume: 5 ml/kg
- Lot nos.: ZP0867 and YH1095, exp. dates: 15 July 2010 and 23 October 2010, respectively.
- Supplier: Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for homogeneity and concentration analyses were collected from the middle stratum of the control formulation and from the top, middle, and bottom strata of each test item formulation used for dose administration. One set of samples from the first, third, fifth, and last batches was subjected to the appropriate analyses. Analyses were conducted by the Analytical Chemistry Department at WIL Research using a validated high performance liquid chromatography method with ultraviolet absorbance detection.

The analyzed dosing formulations were within WIL Research’s SOP range for suspensions (85% to 115%), were homogeneous, and were stable for 10 days at room temperature.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of mating; maximum of 14 days
- Proof of pregnancy: vaginal copulatory plug or the presence of sperm following a vaginal lavage. The day when evidence of mating was identified was termed gestation day 0.
- After successful mating each pregnant female was caged (how): plastic maternity cages with nesting material
- Other: The animals were mated following 14 days of treatment. Pairing was in the home cage of the male.

Duration of treatment / exposure:

Males: Days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses.
Females: Days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-44 doses.
Females: with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 40-52 doses.

Frequency of treatment:

Once daily, at approximately the same time each day.

Duration of test:

28 days

No. of animals per sex per dose:

The low- and mid-dose groups: 10 rats/sex
control and high-dose groups: 15 rats/sex (5 animals were provided a 14-day recovery period)

Control animals:

yes, concurrent vehicle

Details on study design:

All F0 females were allowed to deliver and rear their pups until lactation day 4.
- Age at mating of the mated animals in the study: approximately 14 weeks

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Detailed physical examinations were recorded 1 week prior to initiation of test item administration and weekly thereafter (prior to test item administration during the treatment period). Each male and female was also observed for signs of toxicity approximately 1-2 hours following dose administration. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHT:
Individual female body weights were recorded weekly until evidence of copulation was observed for females selected for pairing. Weekly body weights continued to be recorded for females without evidence of mating and for those females that were not selected for pairing. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible), 1, and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Following mating, food consumption for females with no evidence of mating was measured on a weekly basis until the scheduled euthanasia.

CLINICAL PATHOLOGY: Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 5 animals/sex/group at the scheduled necropsies (study day 28 for males and lactation day 4 for females that were selected for pairing) and from 5 animals/sex in the control and high-dose groups following a 14-day non-dosing post-treatment period (study day 42 for males and study day 53 for females). The same animals selected for FOB and motor activity assessments were selected for clinical pathology assessment. All males (including those not selected for clinical pathology assessments) and the post treatment phase females were fasted overnight prior to blood collection with water available ad libitum. Blood for serum chemistry and hematology was collected from the retro orbital sinus following isoflurane anesthesia. Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing EDTA (hematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry). The following parameters were evaluated:
- Hematology: Total leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Prothrombin time, Activated partial thromboplastin time, Reticulocyte count Percent / Absolute, Mean Platelet Volume, Red cell distribution width, Hemoglobin Distribution Width, Differential leukocyte count, Platelet estimate, Red cell morphology.
- Clinical Chemistry: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio [by calculation], Total bilirubin, Urea nitrogen, Creatinine, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Glucose, Total cholesterol, Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides, Bile acids.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes

Fetal examinations:

PARTURITION:
On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

LITTER VIABILITY AND DEATHS: Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained.

CLINICAL OBERSERVATIONS: Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nursing behavior were recorded.

BODY WEIGHTS: Pups were individually weighed on PND 1 and 4.

SEX DETERMINATION: Pups were individually sexed on PND 0 (if possible), 1, and 4.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit. Statistical tests were performed using WTDMS™ or were conducted by BIOSTAT CONSULTANTS, INC.using SAS version 9.1 (SAS Institute, Inc., 2002 2003), or higher, software.

Indices:

The following offspring viability indices were calculated: Mean Live Litter Size, Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter), Postnatal Survival for All Other Intervals (% Per Litter).




The following parameters were calculated: Mean Live Litter Size, Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter), Postnatal Survival for All Other Intervals (% Per Litter).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: (clinical signs)

Details on maternal toxic effects:
At the time of dose administration, salivation was observed for 5 of 15 females in the 1000 mg/kg/day group; this clinical finding is not considered adverse. Soft stool and yellow and brown material findings noted at 1000 mg/kg/day did not persist into the post-treatment period.

A significantly (p<0.05 or p<0.01) lower mean body weight gain was noted for the 1000 mg/kg/day group females during study days 0-7 and 0-13 (entire pre-mating treatment period). These decrements were due in part to 2 females in this group that lost weight (15 g or 16 g) during study days 0-7. During study days 7-13 (treatment period) and throughout the post-treatment period, mean body weight gains for the 1000 mg/kg/day group were similar to the control group. The effects on mean body weight gain were not of sufficient magnitude to substantially affect mean body weights and were not considered adverse. Mean body weights and body weight gains for the 30 and 100 mg/kg/day groups were comparable to the control group throughout the treatment and post-treatment periods. Mean body weights and body weight gains in the 30, 100, and 1000 mg/kg/day groups were comparable to those in the control group throughout gestation and lactation.

In the liver there was an increased incidence of periportal hepatocellular vacuolation in males administered = 30 mg/kg/day and in the 1000 mg/kg/day group females. This was characterized microscopically as periportal hepatocytes containing minimal to mild numbers of mixed small (microvesicular) and large (macrovesicular) vacuoles in the cytoplasm. There was no evidence of accompanying inflammation, degeneration, or cellular reaction involving hepatocellular, endothelial, Kupffer, or biliary cells. The cytoplasmic vacuolation, presumed lipid, was minimal to mild and not associated with inflammatory or cellular degeneration. Serum chemistry assays helpful in assessing liver toxicity, such as alanine and aspartate aminotransferase, gamma glutamyltransferase, bilirubin, albumin, globulin, cholesterol, and tryglycerides levels, showed no test item-related alterations. Bile acid levels were slightly higher in males and females administered the test item, but these elevations were not statistically significant nor did they show a clear dose relationship. Thus, the minimal to mild periportal hepatocellular vacuolation was not considered to be adverse. Following the 14-day nondosing period, the incidences of periportal hepatocellular vacuolation were reduced in the 1000 mg/kg/day group and/or were comparable to values in the control group.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
Slightly lower mean F1 pup birth weights (PND 1) for the 1000 mg/kg/day group in combination with slightly lower mean body weight gains during PND 1-4 resulted in test item-related lower mean body weights in this group on PND 4. The difference from the concurrent control group for the 1000 mg/kg/day group males on PND 4 was significant (p<0.01). Mean pup body weights for the concurrent control group males and females on PND 1 and 4 were higher than or close to the maximum mean value in the WIL historical control database. However, due to the fact that lower pup birth weights in the 1000 mg/kg/day group were accompanied by lower mean body weight gains during PND 1-4, these differences were considered test item-related. Mean pup body weights and body weight changes in the 30 and 100 mg/kg/day group males and females were comparable to the concurrent control group.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks:

Neonatal toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in litter size and weights

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The study was conducted to evaluate the toxic potential of a tested substance EC 903-161-3 when administered oraly to rats for a period of twenty eight consecutive days at dose levels of 30, 100 and 1000 mg/kg/day. Under the condition of this study, the NOAEL for systemic toxicity was considered to be 100 mg/kg/day based on the clinical findings of soft stool and brown and yellow material around the anogenital area observed at 1000 mg/kg/day. The NOAEL for neonatal toxicity was considered to be 100 mg/kg/day based on lower pup body weights in the 1000 mg/kg/ day group.

Executive summary:

In a guideline repeated dose and reproduction / developmental toxicity screening study (OECD 422, with recovery and endocrine assessments) conducted according to Good Laboratory Practices, the potential toxic effects of a test substance EC 903 -161 -3 were evaluated when administered to rats.

This study was designed to evaluate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development.

The test item, in the vehicle, mineral oil, USP, was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats. The low- and mid-dose groups each consisted of 10 rats/sex and the high-dose group consisted of 15 rats/sex. Dosage levels were 30, 100, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. Ten males/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses. Ten females/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 39-44 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 40-52 doses. The extra 5 rats/sex in the control and high-dose groups were not used for mating and were treated beginning on study day 0; following 28 doses for the males and 40 doses for the females, these animals were assigned to the post-treatment period and remained on study for a 14-day non-dosing period. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and locomotor activity data were recorded for 5males/group following approximately 28 days of dose administration and for 5 females/group on lactation day 4. All F0females were allowed to deliver and rear their pups until lactation day 4. F1clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (hematology and serum chemistry) were performed on 5 F0animals/sex/group at the treatment period and post-treatment period necropsies. F0males were euthanized following completion of the mating period and F0females were euthanized on lactation day 4. Spermatogenic endpoint evaluations were conducted on all males at the treatment and post-treatment period necropsies. Complete necropsies were conducted on all F0animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0animals in the control and high-dose groups that were euthanized at the treatment period necropsy.

All animals survived to the scheduled necropsy. Clinical findings of soft stool and brown and yellow material around the anogenital area were observed for F0animals in the 1000 mg/kg/day group at the weekly detailed physical examinations during the treatment period and/or at approximately 1-2 hours following dose administration. These findings did not persist into the post-treatment period. At the time of dose administration, an increased incidence of salivation was observed for the 1000 mg/kg/day group females when compared with the control group; this finding was not considered adverse. Lower mean body weight gains, in the absence of effects on food consumption, were noted for the 1000 mg/kg/day group females when compared to the control group during study days 0-7, resulting in lower mean body weight gains during study days 0-13; these decrements were not of sufficient magnitude to affect mean body weights. Mean body weights and body weight gains for the 1000 mg/kg/day group males were comparable to the control group throughout the treatment period. During the remainder of the treatment and post-treatment periods (including the gestation and lactation periods for females), mean body weights and body weight gains for the 1000 mg/kg/day group males and females were comparable to the control group. Mean body weights, body weight gains, and food consumption for the 30 and 100 mg/kg/day groups were similar to the control group throughout the study. No test item-related effects were noted during the functional observational battery and locomotor activity assessments. There were no test item-related changes in clinical pathology parameters or gross necropsy observations at any dosage level at the scheduled necropsies during the treatment and post-treatment periods. Mean organ weights in the 30,100, and 1000 mg/kg/day groups were unaffected by test item administration. In addition, mean numbers of corpora lutea, unaccounted-for sites, and implantation sites were similar across groups. In the liver, there was an increased incidence of periportal hepatocellular vacuolation in the 30 mg/kg/day group males and 1000 mg/kg/day group males and females at the end of the treatment period. This cytoplasmic vacuolation was minimal to mild and not associated with inflammatory or cellular degeneration and was not considered to be adverse. Following the 14-day non-dosing period, low incidences of periportal hepatocellular vacuolation were observed in the 1000 mg/kg/day group, indicating recovery and no persistence of the test article-related effect. Mean F1pup body weights in the 1000 mg/kg/day group were 9.2% (males) and 8.3% (females) lower than the control group on PND 1. Slightly lower mean F1body weight gains for this group during PND 1-4 resulted in mean body weights that were 13.8% (males) and 10.8% (females) lower than the control group on PND 4. Mean F1body weights and body weight gains for the 30 and 100 mg/kg/day groups were unaffected by F0maternal test item administration. The mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 30,100, and 1000 mg/kg/day groups were similar to the control group values. No test item-related clinical findings or macroscopic findings for F1pups that were found dead were noted at any dosage level.

Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 100 mg/kg/day based on the clinical findings of soft stool and brown and yellow material around the anogenital area observed at 1000 mg/kg/day. The NOAEL for neonatal toxicity was 100 mg/kg/day based on lower pup body weights in the 1000 mg/kg/day group.