2,2'-methylenebis(4,6-di-tert-butyl-phenyl)-2-ethylhexyl phosphite

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2018 - 16 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Please see materials and methods section.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley Crl:CD®(SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
A sufficient number of male and female Sprague-Dawley Crl:CD®(SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Kent, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 324 to 446g, and were approximately ten weeks old. The females weighed 213 to 288g, and were approximately twelve weeks old.

ENVIRONMENTAL CONDITIONS
Initially, all animals were housed in groups of two or three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. 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. The animals were allowed free access to food and water. A powdered diet (Rodent PMI 5002 (Certified) Ground Diet, BCM IPS Limited, London, UK) was used. 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) except for paired animals and mated females during the final week of gestation and lactation.

Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions
were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan. The animals were randomly allocated to treatment groups using a stratified body weight
randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cages were distributed in dose group columns within the holding rack to minimize the potential of cross contamination of the treated diet.
The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

IN-LIFE DATES: From: 10th September 2018 To: 10th October 2018

Route of administration:

oral: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
For the purpose of this study the test item was incorporated into the diet at concentrations of
2000, 6000 and 20000 ppm as follows:

A known amount of test item was mixed with a small amount of basal laboratory diet until homogeneous in a Robot Coupe Blixer 4 set at a constant speed. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a Hobart H800/U200 mixer.
The stability and homogeneity of the test item dietary admixtures were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the dietary admixtures to be stable for at least twenty-one days at room temperature and at approximately -20 °C. Dietary admixtures were prepared fortnightly and stored at room temperature in labelled double plastic bags, in labelled, covered plastic bins.

Samples were taken of the dietary admixtures on two occasions and analyzed for concentration of HP-10 at Envigo Research Limited, Shardlow, UK, Analytical Services.
The method used for analysis of the dietary admixtures and the results obtained are given in Annex 2. The results indicate that the prepared admixtures were within 93-98% of the nominal concentration.

VEHICLE
No vehicle the test item was mixed with a small amount of diet.

Details on mating procedure:

Pairing of animals within each dose group was undertaken on a one male: one female basis on Day 15 of the study, to produce litters.
Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance, visible nipple counts (male offspring) and clinical signs were also recorded during this period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyical procedure was successfuly validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision. The homogeneity and stability was confirmed for est item in dietary admixtures at nominal concetrations of 2000ppm and 20000 ppm when stored at ambient temperatres and frozen for 21 days.

The mean concentrations of the test item in test formulation analyzed for the study were within +/- 20% of nominal concentrations, confirming accurate preparation.

Duration of treatment / exposure:

Up to eight weeks

Frequency of treatment:

Continuous (dietary admixture)

Details on study schedule:

Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.

Animals received treated diet, according to dose group throughout the study period. Control animals received basal laboratory diet. The first day of dosing was
designated as Day 1 of the study. During the pre-pairing period vaginal smears were performed and assessed for females.

On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance, visible nipple
counts (male offspring) and clinical signs were also recorded during this period.

On Day 4 post partum, where possible, blood sampling was performed on two randomly allocated offspring from each litter in order to obtain serum samples.

The male dose groups were killed and examined macroscopically on Day 32 or 33.

On Day 13 post partum, where possible, blood sampling to produce serum samples for assessment of thyroid hormones (T4) was performed on two randomly selected
offspring (one male and one female) per litter. Where possible, a further two randomly selected offspring (one male and one female) per litter were sampled to
produce serum samples for possible TSH analysis. Thyroid/parathyroid samples were also retained from one male and one female from each litter where litter sizes allowed. All surviving offspring were killed and examined externally; where external observations were detected an internal necropsy was performed.

All females were sacrificed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all females in the morning of the day of
necropsy. Any female which did not produce a pregnancy was also sacrificed and examined macroscopically around the same time as littering females. In addition,
blood samples to produce two serum samples were taken from all adult animals at termination. One of the blood samples from all adult males and Day 13 offspring
were analyzed for Thyroxine (T4).

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

2 000 ppm

Remarks:

Equivalent to a 118.7 mg/kg bw/day for males, 137.9 mg/kg bw/day for females during maturation, 149.6 mg/kg bw/day for females during gestation and 285.1 mg/kg bw/day for females during lactation.

Dose / conc.:

6 000 ppm

Remarks:

Equivalent to 370.0 mg/kg bw/day for males, 399.3 mg/kg bw/day for females during maturation, 448.3 mg/kg bw/day for females during gestation and 839.5 mg/kg bw/day respectively for females during lactation.

Dose / conc.:

20 000 ppm

Remarks:

Equivalent to 1212.6 mg/kg bw/day for males, 1351.1 mg/kg bw/day for females during maturation, 1508.4 mg/kg bw/day for females during gestation and 2966.4 mg/kg bw/day for females during lactation.

No. of animals per sex per dose:

Ten

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

Clinical signs, body weight development, food and water consumption were monitored during the study.

Oestrous cyclicity (parental animals):

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all females through prepairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females.

Litter observations:

STANDARDISATION OF LITTERS
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post
partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were
calculated retrospectively from this data)

PARAMETERS EXAMINED
- Daily clinical observations were undertaken on all live offspring, together with litter size, offspring weights and assessment of anogenital distance and visible nipple count (male offspring only).
- Blood samples were taken at termination from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis Thyroxine (T4).



GROSS EXAMINATION OF DEAD PUPS:
Yes - external and internal abnormalities.

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

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

Statistics:

Please see methods section.

Reproductive indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = number of animals mated/ number of animals paired x 100

Pregnancy Index (%) = number of pregnant females/ number of animals mated x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = number of females delivering live offspring/ number of pregnant females x 100

Offspring viability indices:

Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = number of offspring alive on Day 1/ number of offspring born x 100

Viability Index 1 (%) = number of offspring alive on Day 4/ number of offspring alive on Day 1 x 100

Viability Index 2 (%) = number of offspring alive on Day 13/ number of offspring alive on Day 4 x 100

Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post partum.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse clinical signs detected in treated animals.
One male fed diet containing 20000 ppm had a dark eye between Days 26 and 33 and one female from this dosage group had generalized fur loss between Days 39 and 52. One male fed diet containing 6000 ppm had scab formation between Days 15 and 32 and one female fed diet containing 2000 ppm had corneal opacity between Days 42 and 51. These observations were seen in isolation and were considered to be incidental and unrelated to treatment. One female fed diet containing 2000 ppm had pilo-erection on Day 41. This was observed around the time of littering for this female and was therefore unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse effect in body weight development was evident in treated males.
No adverse effect in body weight development was evident in treated females during maturation, gestation or lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No adverse effect on food consumption was evident in treated males.
No adverse effect on food consumption was evident in treated females during maturation, gestation or lactation.

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

no effects observed

Description (incidence and severity):

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic abnormalities detected.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic abnormalities detected.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Assessment.
Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 2000, 6000 or 20000 ppm.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 2000, 6000 or 20000 ppm, with all females showing regular cycling. Assessment of estrous cycles at termination did not indicate any obvious effect of treatment at 2000, 6000 or 20000 ppm.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, as assessed by the number of paired animals that mated, was unaffected by treatment at dosages of 2000, 6000 or 20000 ppm. All animals mated within the first four days after pairing.

No treatment-related effects were detected in mating performance or in fertility. Gestation lengths were essentially similar to control.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 20 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical sign findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 2000, 6000 or 20000 ppm.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Offspring body weight, offspring body weight gains and litter weights on Days 1, 4 and 13 post partum were comparable to controls.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Male offspring body weights on Day 7, body weight gains between Days 4 and 7 and cumulative body weight gains between Days 1 and 7 were statistically significantly increased (p<0.05) in litters from females fed diet containing 2000 ppm. An increase in body weight /body weight gain is considered not to represent an adverse effect of treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect
of maternal treatment on offspring development at 2000, 6000 or 20000 ppm.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic abnormalities detected.

Of the litters born, litter size at birth and subsequently on Days 1, 4 and 13 post partum were comparable to controls.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

ca. 20 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The oral administration of HP-10 to rats, at dietary concentrations of 2000, 6000 and 20000 ppm did not result in any treatment-related effects. Within the confines of this study, the No Observed Effect Level (NOEL) for adult toxicity was considered to be 20000 ppm (equivalent to a mean achieved dosage of 1212.6 mg/kg bw/day for males and 1351.1 mg/kg bw/day for females during maturation).
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 20000 ppm.

Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, to evaluate some endocrine disruptor relevant endpoints, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).

This study was also designed to be compatible with 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 continuous dietary admixture to three groups, each of ten male and ten female Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, for approximately five weeks for males and eight weeks for females (including a two week pre-pairing phase, pairing, gestation and lactation) at dietary concentrations of 2000, 6000 and 20000 ppm (equivalent to a mean achieved dosage of 118.7, 370.0 and 1212.6 mg/kg bw/day respectively for males, 137.9, 399.3 and 1351.1 mg/kg bw/day respectively for females during maturation, 149.6, 448.3 and 1508.4 mg/kg bw/day respectively for females during gestation and 285.1, 839.5 and 2966.4 mg/kg bw/day respectively for females during lactation). A control group of ten males and ten females were treated with basal laboratory diet over the same period.

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with recording of litter size, offspring weights, ano-genital distance and visible nipple count (male offspring only).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were terminated on Day 32 or 33, followed by the termination of all surviving offspring and adult females on Days 13 and 14post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Results…….

Adult Responses

Mortality

There were no unscheduled deaths.

Clinical Observations

There were no adverse clinical signs detected in treated animals.

Body Weight

No adverse effect in body weight development was evident in treated males.

No adverse effect in body weight development was evident in treated females during maturation, gestation or lactation.

Food Consumption

No adverse effect on food consumption was evident in treated males.

No adverse effect on food consumption was evident in treated females during maturation, gestation or lactation.

Water Consumption

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Reproductive Performance

Estrous Cycle

There was no effect of treatment with the test item at any dietary concentration on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.

 

Mating

There was no effect of treatment on mating performance.

Fertility

There were no treatment-related effects in conception rates for test item-treated animals in relation to controls.

Gestation Length

There were no differences in gestation lengths in animals receiving the test item when compared with controls.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no detrimental effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 2000, 6000 or 20000 ppm.

Offspring Growth and Development

There was no effect of treatment with the test item indicated by clinical signs, offspring body weight or body weight gain, visible nipple count in male offspring on Day 13post partum, or ano-genital distance at 2000, 6000 or 20000 ppm.

Pathology

Necropsy

Offspring

Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any obvious effect of maternal treatment at 2000, 6000 or 20000 ppm.

Adults

There were no toxicologically significant macroscopic abnormalities detected.

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 2000, 6000 or 20000 ppm.

 

Organ Weights

Assessment of male organ weights did not indicate any treatment-related effects at 2000, 6000 or 20000 ppm.

Histopathology

There were no treatment-related microscopic abnormalities detected.

Conclusion

The oral administration of HP-10 to rats, at dietary concentrations of 2000, 6000 and 20000 ppm did not result in any treatment-related effects. Within the confines of this study, the No Observed Effect Level (NOEL) for adult toxicity was considered to be 20000 ppm (equivalent to a mean achieved dosage of 1212.6 mg/kg bw/day for males and 1351.1 mg/kg bw/day for females during maturation).

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 20000 ppm.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed