Phosphoric acid, mono- and di-C16-18-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test guideline (migrated information

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The oral administration of the test material to pregnant rats by oral gavage during gestation at nominal dose levels of 100, 300 and 1000 mg/kg bw/day (analytical mean dosages of 110.9, 263.2 and 712.9 mg/kg bw/day), resulted in the early termination of one female at 1000 mg/kg bw/day. This death was considered most likely to be due to the physical nature of the test material formulation and not related to systemic toxicity. 

The ‘No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be 1000 mg/kg bw/day (actual mean dosage of 712.9 mg/kg bw/day).

There were no treatment-related changes detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day (actual mean dosage of 712.9 mg/kg bw/day).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 July 2017 to xxxx

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147 (24 November 2000)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Physical State/Appearance: White waxy solid
Purity: 100%, UVCB substance
Storage Conditions: Room temperature in the dark

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(R) (SD) IGS BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
A total of ninety-six time-mated female Sprague-Dawley Crl:CD¿ (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 196 to 284g on either Day 0 or 1 of gestation.

The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes. The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet) 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. 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. 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 mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 +/- 3 deg.C and 50 +/- 20% respectively.

The animals were weighed upon arrival and randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories. In addition, a color-coded cage card was prepared with details of test material, study number, dose level, animal number, route of administration and Study Director.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

The test material was heated to approximately 85 deg.C to form a solution in Arachis oil at 6 mL/kg and to ensure the test material formulations remained in a dosable state for the duration of the dosing period. During the dosing procedure the test material formulations and control vehicle were kept in a warming bath at approximately 40 deg.C.

The test material was administered daily, from Day 5 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone. The volume of test and control material administered to each animal was based on the most recently recorded body weight.

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Time-mated females were obtained from Charles River (UK) Limited prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation by the supplier.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Days 5 to 19 after mating

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were chosen in consultation with the Study Monitor for the Sponsor and were based on previous toxicity data. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material, and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man.

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

For the purpose of the study the test material was prepared at the target concentrations as a straw coloured solution in Arachis Oil after heating to 85 deg.C and then mixed. The stability and homogeneity of the test material formulations were determined by Envigo Research Limited Analytical Services. Formulations were prepared twice weekly and stored at approximately +4 deg.C in the dark. Test material formulations stored under the storage conditions were reheated and mixed by shaking the formulation well to get the formulation back into solution prior to dosing.

Samples were taken of test material formulations and were analyzed for achieved concentration on five occasions at Envigo Analytical Laboratory. The analytical results showed the dose formulation was not optimal for analytical purposes due to the physical properties of the formulation at room temperature, as variable ranges of dose concentration were reported. Whilst uniform solutions of the test material were observed at dose formulation when the test material was melted at 85 deg.C and mixed with warm vehicle, solidification of the test material formulation was observed after a short period of time once the temperature of the formulations declined. Due to the nature of the formulations all attempts were made to ensure that dose administration was performed within 10 to 15 minutes of dose formulation/reformulation. Dose levels in the report reflect analytical chemistry figures for achieved dose concentration, however it is likely that animals, particularly at the highest dose level were exposed at levels above the recorded achieved concentration.

Maternal examinations:

Animals were checked twice daily, early and late during the working period, for morbidity and mortality.

Clinical Observations:
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing.

Body Weight:
Individual body weights were recorded on Day 3 of gestation (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20 of gestation).

Food Consumption:
Food consumption was recorded for each surviving individual animal on Days 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption:
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

Necropsy
All surviving animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.

Ovaries and uterine content:

The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Fetal examinations:

Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were placed in Bouin’s fixative, subsequently transferred to distilled water, and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were placed into 70% Industrial Methylated Spirit (IMS) in distilled water, were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and subsequent storage.

Statistics:

The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.

All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.

Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p=0.05 (not significant)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical observations:
Three females treated with 1000 mg/kg bw/day were observed with noisy respiration for 1-3 days of treatment. One of these females also showed staining around the mouth and snout and pilo-erection during the final two days on study. No such effects were evident in females treated with 300 or 100 mg/kg bw/day.

Not considered to affect NOAEL.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One high dose female was killed in extremis on Day 17 of gestation. There were no further unscheduled deaths on the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse effect was detected in body weight or body weight gains.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect detected on food consumption.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No adverse effect on water consumption was detected.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

One surviving female treated with 1000 mg/kg bw/day had gaseous distension in the stomach and intestines and white solid material in the stomach. No such effects were evident in females treated with 300 or 100 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

Dead fetuses:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

Key result

Dose descriptor:

NOAEC

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

Analytical mean dosage of 712.9 mg/kg bw/day

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

necropsy findings

number of abortions

pre and post implantation loss

total litter losses by resorption

water consumption and compound intake

Remarks on result:

other: Nominal was considered more reliable due to problems with solidification of the test substance.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in fetal external examinations.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in fetal external examinations.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in fetal external examinations.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in fetal external examinations.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in fetal external examinations.

External malformations:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in fetal external examinations.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No treatment-related adverse effects on skeletal development were detected or in the type and incidence of visceral findings in fetuses from females treated with 100, 300 or 1000 mg/kg bw/day.

Visceral malformations:

no effects observed

Description (incidence and severity):

No treatment-related adverse effects on skeletal development were detected or in the type and incidence of visceral findings in fetuses from females treated with 100, 300 or 1000 mg/kg bw/day.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

Actual mean dosage of 712.9 mg/kg bw/day

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Developmental effects observed:

not specified

Due to the physical nature of the test material, the preparation of the dose formulation and analytical sampling were challenging and this was reflected in the variable results for analytical concentrations during the study in relation to the nominal dose concentrations.

Formulation records indicate that the correct amount of test material and vehicle were used during dose preparation at each concentration on every occasion. The test material was heated to 85 deg.C to produce a liquid state and then mixed with the required amount of warm vehicle (Arachis oil) to produce a dosable liquid formulation. These formulations were sub-sampled for concentration analysis. These smaller sub-samples generally solidified during transportation to the analytical chemists and were therefore heated to 50 deg.C (as per the validated method) for between 30 to 45 minutes in order to return the samples to a liquid state suitable for the analysis procedure.

The solidification of the samples as they cooled during the sampling process probably resulted in losses of test material on the sampling equipment and it is possible that not all the Test material had been fully dissolved back into the formulation samples.

On the basis of the analytical results recorded, the calculated analytical mean dose levels for the low, intermediate and high dosage groups were 110.9, 263.2 and 712.9 mg/kg bw/day respectively. However, it is considered, based upon the nature of the test material, that the animals on study would have received a higher dose concentration than the analytical results imply, and the actual dosage received was closer to the intended nominal dosage.

Conclusions:

The oral administration to pregnant rats by oral gavage during gestation at nominal dose levels of 100, 300 and 1000 mg/kg bw/day (analytical doses of 110.9, 263.2 and 712.9 mg/kg bw/day), resulted in the early termination of one female at 1000 mg/kg bw/day. This death was considered most likely to be due to the physical nature of the test material formulation and not related to systemic toxicity. The ‘No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be 1000 mg/kg bw/day (analytical mean dose of 712.9 mg/kg bw/day).

There were no treatment-related changes detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day (analytical mean dose of 712.9 mg/kg bw/day).

Executive summary:

The study was performed to investigate the effects of the test material on embryonic and fetal development following repeated administration by gavage to the pregnant female rat during gestation including the period of organogenesis.

The test material was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at nominal dose levels of 100, 300, and 1000 mg/kg bw/day (analytical mean doses of 110.9, 263.2 and 712.9 mg/kg bw/day). A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined in detail for skeletal development and the remaining half were subjected to detailed visceral examination.

One high dose female was killed in extremison Day 17 of gestation. There were no further unscheduled deaths on the study.

Three females treated with 1000 mg/kg bw/day were observed with noisy respiration for 1-3 days of treatment. One of these females also showed staining around the mouth and snout and pilo-erection during the final two days on study. No such effects were evident in females treated with 300 or 100 mg/kg bw/day.

No adverse effect was detected in body weight or body weight gains, food consumption and water consumption.

One surviving female treated with 1000 mg/kg bw/day had gaseous distension in the stomach and intestines and white solid material in the stomach. No such effects were evident in females treated with 300 or 100 mg/kg bw/day.

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

No treatment-related effects were detected in fetal external examinations. No treatment-related adverse effects on skeletal development were detected or in the type and incidence of visceral findings in fetuses from females treated with 100, 300 or 1000 mg/kg bw/day.

In conclusion, the oral administration of the test material to pregnant rats by oral gavage during gestation at nominal dose levels of 100, 300 and 1000 mg/kg bw/day (analytical mean doses of 110.9, 263.2 and 712.9 mg/kg bw/day), resulted in the early termination of one female at 1000 mg/kg bw/day. This death was considered most likely to be due to the physical nature of the test material formulation and not related to systemic toxicity. The ‘No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be 1000 mg/kg bw/day (analytical mean dose of 712.9 mg/kg bw/day).

There were no treatment-related changes detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day (analytical mean dose of 712.9 mg/kg bw/day).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

The ‘No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be 1000 mg/kg bw/day (actual mean dosage of 712.9 mg/kg bw/day).

There were no treatment-related changes detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day (actual mean dosage of 712.9 mg/kg bw/day).