L-97-034-PB

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10th October 2018 - 19th March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pentaerythritol mixed esters with 2-methyl butyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid, n-decanoic acid

Cas Number:

141686-49-9

IUPAC Name:

Pentaerythritol mixed esters with 2-methyl butyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid, n-decanoic acid

Constituent 2

Reference substance name:

Trimethylol propane mixed esters with 2-methyl butyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid, n-decanoic acid

Cas Number:

141686-50-2

IUPAC Name:

Trimethylol propane mixed esters with 2-methyl butyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid, n-decanoic acid

Test material form:

liquid

Details on test material:

Identification: Hatcol ® 1772
Appearance/physical state: Clear colorless liquid
Purity: 100% (no solvent present)
Composition: 70% pentaerythritol esters described by CAS# 141686-49-9; 30% trimethylolpropane esters described by CAS# 141686-50-2

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Strain/Species: Crl:CD(SD) rat
Supplier: Charles River (UK) Ltd
Number of animals ordered: 44 males and 48 females. Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization: Males: eight days prior to the commencement of treatment. Females: 22 days prior to the commencement of treatment.
Age of the animals at the start of treatment: Males - 71 to 78 days old; Females - 85 to 92 days old.
Weight range of the animals at the start of treatment: Males - 343 to 416 g; Females - 247 to 310 g.

ALLOCATION AND IDENTIFICATION
Allocation: On arrival and non-selective allocation to cages. Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study. On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced to ensure variations in body weight of animals did not exceed +/- 20% of the mean for each sex.

Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a microchip before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as
well as the identity of the occupant(s).

ANIMAL REPLACEMENT
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Two females with irregular estrous cycles were rejected during the acclimatization period and were replaced with spare animals of suitable weight from the same batch.

ENVIRONMENTAL CONTROL
Rodent facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%. There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

ANIMAL ACCOMMODATION
Cages: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

ENVIRONMENTAL ENRICHMENT
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Polycarbonate shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.
Nesting material: From Day 20 after mating and throughout lactation, paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

DIET SUPPLY
Diet: SDS VRF1 Certified pelleted diet. A sample (250 g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30ºC) until finalization of
the report. Samples were discarded after finalization of the report. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: Non-restricted.

WATER SUPPLY
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted.

SUPPLIER CERTIFICATES OF ANALYSIS
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier. Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding, Aspen chew blocks and polycarbonate shelters. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Method of preparation: The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. A series of formulations at the required concentrations were prepared in ascending order by dilution of individual weighings of the test item.
Frequency of preparation: Formulations were prepared and used within the known stability period.
Storage of formulation: Refrigerated (2 to 8°C).
Test item accounting: Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Details on mating procedure:

MATING PROCEDURE

Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

FORMULATION ANALYSIS
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.

The homogeneity and stability was confirmed for Hatcol® 1772 in arachis oil formulations at nominal concentrations of 10 mg/mL and 250 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (15 to 25ºC) for 1 day and refrigerated storage (2 to 8ºC) for up to 15 days.

The mean concentrations of Hatcol® 1772 in test formulations analyzed for the study were within 10% of nominal concentrations, confirming accurate formulation. The difference from mean remained within 2%, confirming precise analysis. The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical methodology.

Duration of treatment / exposure:

DURATION OF TREATMENT
Males: Two weeks pre-pairing up to necropsy after minimum of four weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Once daily at approximately the same time each day.

Details on study schedule:

Study initiation (Study Plan signed by Study Director): 10 October 2018
Experimental start date (Pre-study chemistry): 21 October 2018
Animal arrival - Males: 5 December 2018; Females: 21 November 2018
Estrous cycle evaluation commenced: 29 November 2018
Treatment commenced: 13 December 2018
F0 pairing commenced: 27 December 2018
F0 necropsy - Males: 21 January 2019; Females: 31 January to 6 February 2019
Experimental completion date (Bioanalytical Phase, last date of analysis of samples): 19 March 2019

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10 male and 10 female animals per dose

Control animals:

yes, concurrent vehicle

Details on study design:

RATIONALE FOR DOSE SELECTION
The doses used in this study (0, 100, 300 and 1000 mg/kg/day) were selected in conjunction with the Sponsor.

A 28-day repeat dose oral (gavage) toxicity study with L-97-034-PB (the R&D name of the test item) had been conducted (Ref: SPL Project Number: 334/122). The dose levels selected for that study were 0, 150, 500 and 1000 mg/kg/day. One control group and three treated groups were tested, each group consisting of five male and five female Sprague-Dawley Crl:CD®BR strain rats. The following parameters were evaluated: clinical signs, behavioral assessments in the arena, motor activity, forelimb/hindlimb grip strength, sensory reactivity, body weight, food consumption, visual water consumption, clinical pathology, macropathology and organ weights. Histopathology was performed on a selection of tissues but for the liver, spleen and kidney these tissues were examined for all animals.

In the females, treatment resulted in slightly reduced food consumption at 1000 mg/kg/day, increased liver weights at 500 or 1000 mg/kg/day and pallor of the liver in two females given 1000 mg/kg/day and in one female given 500 mg/kg/day. Males treated at 500 or 1000 mg/kg/day showed increased kidney weights and males given 1000 mg/kg/day showed speckled kidneys at macroscopic examination. Histopathological examination of the kidneys revealed globular accumulations of eosinophilic material in the renal proximal tubular epithelium of males treated with 150, 500 or 1000 mg/kg/day. This was classified as ‘hydrocarbon nephropathy’ which is peculiar to the male rat and occurs in response to treatment with certain hydrocarbons (female rats and other species do not develop ‘hydrocarbon nephropathology’ and the effect is not indicative of hazard to human health). The study concluded that a clear No-Observed-Effect level (NOEL) was not obtained for males, because of the increased liver weight and hydrocarbon nephropathy, and the NOEL for females was considered to be 500 mg/kg/day because of the increased liver weight (but with no concomitant histopathology). Given that no effects were reported in the reproductive organs in the 28-day study and that the food consumption effect in the females was only ‘slight’ the high dose level for this screening study could be 1000 mg/kg/day. The intermediate and low doses selected were 300 and 100 mg/kg/day, respectively, to provide a geometric spacing of the dose levels.

Positive control:

Not included

Examinations

Parental animals: Observations and examinations:

SERIAL OBSERVATIONS

CLINICAL OBSERVATIONS
Clinical observations are presented for each animal that showed signs, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable. There were no signs associated with dosing observed and therefore these data are not presented.

BODY WEIGHT
Group mean values and SD were calculated from individual body weight data on each recorded occasion. For the offspring, litter mean body weight (+ SD) was calculated separately for males and females and the group mean values derived from the individual litter values. Group mean weight changes were calculated from the weight changes of individual animals surviving the specified period. Offspring body weight change was calculated relative to Day 1 of age. Body weights were plotted graphically with respect to the start of dosing or the start of the relevant period.

FOOD CONSUMPTION
Group mean food consumptions and standard deviations were derived from unrounded cage values. The column header day numbers represent the days on which the full feeder and empty feeder were recorded.

Oestrous cyclicity (parental animals):

The incidence and percentage females showing the following classifications of estrous cycles before treatment commenced are presented:

Regular: All observed cycles of 4 or 5 days
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus

Vaginal smearing prior to termination is presented in terms of numbers of females that showed estrus during this period and the cycle stage at termination.

Sperm parameters (parental animals):

Not determined

Litter observations:

RECORDS MADE DURING LITTERING PHASE
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Ano-genital distance: Day 1 of age - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.

Postmortem examinations (parental animals):

METHOD OF KILL
All adult animals: Carbon dioxide asphyxiation.
Sequence: To allow satisfactory inter-group comparison.

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Postmortem examinations (offspring):

METHOD OF KILL
Offspring - selected for thyroid hormone sampling on Day 4 and 13 of age: Decapitation.
Offspring - remaining: Intraperitoneal injection of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison.

Premature deaths: Where possible, a fresh macroscopic examination (external) with an assessment of stomach for milk content. Abnormalities were retained in appropriate fixative.
F1 offspring on Day 4 of age: Externally normal offspring were discarded without examination. Externally abnormal offspring were subject to an external macroscopic examination and retained pending possible future examination.
F1 offspring on Day 13 of age: All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormalities were retained in appropriate fixative. Thyroid glands were preserved from two offspring per litter, one male and one female in each litter, where possible.

Statistics:

See below

Reproductive indices:

See below

Offspring viability indices:

See below

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Details on results (P0)

See below

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Mating performance, fertility, litter size and the post natal survival, growth and development of the offspring was unaffected by treatment.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Details on results (F1)

See below

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See 'Remarks'

Remarks on result:

other:

Remarks:

Litter size and offspring survival, clinical condition, body weight/gain, ano-genital distance, nipple counts and macropathology were unaffected by treatment.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

F0 RESPONSES

Clinical observations

There were no clinical signs observed that were considered to be related to treatment with Hatcol® 1772.

There were no dosing signs observed, therefore no data are presented.

One Control male (Animal No. 5) was found dead on Day 33 of treatment and the macroscopic examination revealed a perforated esophagus with a dark area present (which correlated microscopically with inflammation and necrosis), thin and clear thoracic fluid, adhesions (involving multiple organs) and a thickened pericardium. The cause of death was a dosing injury.

One Control female (Animal No. 109) was euthanized for welfare reasons on Lactation Day 10 due to general poor clinical condition; clinical signs prior to dispatch consisted of thin build, abnormal gait, irregular breathing and an aqueous brown discharge from the anus. Dark areas in the mammary tissue and nipples, pale areas on the heart, pale areas in the lungs (which correlated microscopically with alveolar macrophage aggregates), and an enlarged pale adrenal gland (which correlated microscopically with adrenal necrosis) were observed on macroscopic examination.

One female given 1000 mg/kg/day (Animal No. 133) was found dead on Day 20 of gestation, the animal had no clinical signs recorded prior to death and had no macroscopic findings at necropsy. The cause of death or deterioration in clinical condition of this animal was undetermined.

Body Weight

The overall body weight gain during the treatment period (Day 1-40) was marginally low in males receiving 100, 300 or 1000 mg/kg/day of Hatcol® 1772, when compared with the controls (88%, 86% and 86% of Control for Groups 2, 3 and 4, respectively). Group mean body weight gain for females prior to pairing (Day 1-15) was low in females receiving 300 mg/kg/day (58% of Controls) and statistically significantly low in females receiving 1000 mg/kg/day (33% of Controls).

The body weight gains during Gestation (Day 0-20) and lactation were similar to the Controls in females that received 100, 300 or 1000 mg/kg/day.

Food consumption

Group mean food consumption of males and females treated with Hatcol® 1772 was generally similar to the Controls, prior to pairing (males and females), during gestation and lactation.

Estrous Cycles, Pre-Coital Interval, Mating Performance, Fertility and Gestation Length and Gestation Index

Estrous cycles, pre-coital interval, mating performance and fertility was considered unaffected by treatment with Hatcol® 1772, when compared with the Controls.

Three females in the Control group and two females receiving 300 mg/kg/day had an irregular cycle during treatment, however once in pairing all females mated at the first possible opportunity. One Control female was acyclic during treatment. Most females in all groups were in diestrus at termination.

Gestation length was within the expected time frame of 22-23 days for all females, with the exception of one female receiving 100 mg/kg/day. Gestation length and gestation index were considered to be unaffected by treatment at any dose level.

Organ weights

Group mean absolute and adjusted organ weights for males and females that received doses of 100, 300 or 1000 mg/kg/day were generally similar to those of the Controls and considered to be unaffected by treatment.

Macropathology

At macroscopic examination, six males given 1000 mg/kg/day and one male given 300 mg/kg/day had pale kidneys. There were no other macroscopic findings in males treated with Hatcol® 1772 for six weeks that were related to treatment.

Histopathology

Treatment related findings

Kidneys

Changes related to treatment with Hatcol® 1772 were seen in the kidneys (hyaline droplet accumulation) of males given 300 or 1000 mg/kg/day that showed macroscopic abnormalities at necropsy.

Incidental findings

A mammary adenocarcinoma was observed on microscopic examination of one female (Animal Number 136) given 1000 mg/kg/day and correlated with a cervical mass observed at macroscopic examination. This was considered to be incidental as this can occur in animals of this age (Kuzutani, K et al., 2012). The incidence and distribution of all other findings were considered to be unrelated to treatment.

F1 RESPONSES

Offspring clinical signs

There were no clinical signs considered to be related to parental treatment.

Litter Size, Sex Ratio and Survival Indices

The mean numbers of implantations and litter size were slightly low at 1000, 300 or 100 mg/kg/day compared with concurrent control (Implantations: 14.2, 14.6 and 15.1 at 1000, 300 or 100 mg/kg/day, respectively. Litter size: 13.9, 13.9 and 13.7 at 1000, 300 or 100 mg/kg/day, respectively). No effect of parental treatment was inferred as total litter size was within the historical control data (HCD) range in all treated groups (HCD: 26 studies: minimum of 13.7 and maximum of 16.3).

The live birth index was slightly low in all treated groups compared with the concurrent control but there was no dose response (92.1, 87.4 and 93.7% at 1000, 300 or 100 mg/kg/day, respectively), and as all values in the high dose group were within the HCD range, no effect of parental treatment was inferred (HCD: 26 studies: minimum of 92% and maximum of 100%).

The viability and lactation indices were unaffected by parental treatment. The sex ratio was unaffected by treatment.

Ano-Genital Distance

There was no conclusive effect on ano-genital distance in the offspring of parents treated with Hatcol® 1772.

Nipple Counts

No nipples were observed in male offspring of parents treated with Hatcol® 1772.

Offspring Body Weight

Mean offspring body weights on Day 1 of age and offspring body weight gain up to Day 13 of age were unaffected by parental treatment.

Offspring Macropathology

There were no macropathology findings in the offspring considered to be related to parental treatment. No milk in stomach was a common finding among decedent offspring.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive/developmental toxicity and endocrine disruptor relevant endpoints was 1000 mg/kg/day.

Executive summary:

The purpose of this study was a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, by oral gavage administration of the test item, Hatcol® 1772 (an industrial chemical), to Sprague Dawley rats for at least four weeks.

There was considered to be no effect of treatment on serum T4 levels in F0 males or male and female offspring at Day 13 of age.

There were no deaths among the F0 males and females or changes in clinical condition related to treatment with Hatcol® 1772.

Body weight gains of F0 males were marginally low in all treated groups and weight gain of females receiving 1000 mg/kg/day was low before pairing.

Food consumption was unaffected by treatment.

Estrus cycles, mating performance, fertility and gestation length and index were unaffected by treatment.

Organ weights were unaffected by treatment.

One male given 300 mg/kg/day and six males given 1000 mg/kg/day had pale kidneys at macroscopic examination and this correlated with hyaline droplet accumulation microscopically.

Litter size and offspring survival, clinical condition, body weight/gain, ano-genital distance, nipple counts and macropathology were unaffected by treatment.

Based on the results of this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive/developmental toxicity and endocrine disruptor relevant endpoints was 1000 mg/kg/day.