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Diss Factsheets

Toxicological information

Toxicity to reproduction

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Administrative data

screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
of 1995
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
4-(2-{4-[(diphenoxyphosphoryl)oxy]phenyl}propan-2-yl)phenyl diphenyl phosphate; 4-{2-[4-({[4-(2-{4-[(diphenoxyphosphoryl)oxy]phenyl}propan-2-yl)phenoxy](phenoxy)phosphoryl}oxy)phenyl]propan-2-yl}phenyl diphenyl phosphate

Test animals

Details on test animals or test system and environmental conditions:
- Wistar rats, strain: Wistar HAN(TM):HsdRCCHHan(TM):WIST with appropriate range of bodyweight at study start.
- Source: Harlan Laboratories UK Ltd.
- Age at study initiation (treatment start): Ca. 12 weeks.
- Weight at study initiation (treatment start): Males: minimum 312 g, maximum 364 g,
Females: minimum 196 g, maximum 248 g.
- Housing
all animals, before pairing: In groups of 5 by sex in solid floor propylene cages with stainless steel mesh lids.
during pairing (1 male+1 female/cage): In propylene grid-floor cages suspended over absorbent paper-lined trays.
after pairing: Males returned to their initial cages,
during gestation and lactation: Females housed individually in solid floor propylene cages with stainless steel mesh lids.
- Bedding (all animals, except during pairing): Softwood flake bedding (Datesand Ltd, Cheshire, UK).
- Environmental enrichment,
except during gestation/lactation: Wooden chew blocks and cardboard fun tunnels (Datesand Ltd, Cheshire, UK).
- Diet (ad libitum): Commercially available pelleted diet:
Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories UK Ltd.
- Water (ad libitum): Mains drinking water
- Acclimation period: 14 days before treatment start

The animal room was maintained at (target ranges for temperature and relative humidity):
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 55 ± 15%
- Photoperiod (low intensity fluorescent lighting): 12 h day / 12 h night
- Rate of air exchange: At least 15 changes/h
Slight deviations from these targets were considered not to have affected the purpose or integrity of the study.

- Duration of Pre-pairing treatment: 14 days
- Duration of Pairing (until conception): 1 to 5 days
- Duration of Gestation: 22 to 23.5 days

Administration / exposure

Route of administration:
oral: gavage
polyethylene glycol
Details on exposure:
Treatment of parental animals by oral gavage administration. Test substance was not directly administered to F1 animals, as these pups were sacrificed on day 5 post partum, i.e. day 5 of lactation.
Details on mating procedure:
- Male/female ratio per cage: 1/1
- Length of cohabitation: 1 to 5 days, as by then 10 females/group had shown evidence of copulation.
- Proof of pregnancy: Formation of vaginal plug in situ or sperm in vaginal smear referred to as day 0 of gestation.
During cohabitation, cage tray-liners were checked each morning for the presence of ejected copulation plugs and females for pregnancy.

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
- Chemical analysis of test material formulations by high performance liquid chromatography (HPLC) using an external standard technique.
- Concentrations (verified at weekly intervals) of the test material formulations were confirmed at each dose level and homogeneity and stability at
the low and top dose level.
- Chemical analysis confirmed that the prepared formulations were within ± 3% of the corresponding nominal concentration.
Duration of treatment / exposure:
- Treatment period, parental males: Once daily until termination on Day 43 of the study.
Treatment period, parental females: Up to 46 days (from 14 days prior to mating to Day 5 of lactation)
- Frequency of treatment: Once daily (except for females during parturition where applicable)
- Duration of test, parental males: 43 days
Duration of test, parental females: From 14 days prior to mating to Day 5 post partum (Day 5 of lactation)
- Duration of pre-pairing treatment: 14 days

Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: Ca. 14 to 15 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control:
Not included in the study.


Parental animals: Observations and examinations:
Clinical observations performed and frequency:
- Clinical signs: At least three times a day (just before and at regular intervals after administration)
- Body weight, Males: Once a week
Body weight, Females: Once a week for pre-mating and mating period, on Days 0, 7, 14, 20 of gestation and on Days 1 and 4 of lactation.
- Food consumption, Males: Once a week during the pre-mating period and after the mating period.
Food consumption, Females: Once a week during the pre-mating period, for Days 0-7, 7-14 and 14-20 of gestation and for Days 1-4 of lactation.
- Food efficiency: (bodyweight gain/food intake) was calculated retrospectively during pre-maiting and Weeks 1 and 2 of gestation.
- Water consumption: Daily (visual inspection of water bottles for overt changes for each cage group)
Oestrous cyclicity (parental animals):
Frequency of vaginal estrus was determined during the mating period until the day of confirmed copulation.
Sperm parameters (parental animals):
Parameters examined in male parental animals: testis weight, epididymis weight
In addition the degree of the presence of sperm in vaginal smears was assessed by assignment of 3 grades as part of the copulation check.
Litter observations:
STANDARDISATION OF LITTERS: Not performed. The study ended on day 5 post partum.

- No. of pups alive and dead on day 0 post partum, afterwards daily recording of number of live offspring
- Sex ratio (% males) for each litter on days 0 (at birth) 1 and 4
- Clinical signs at least once daily from birth to day 5
- Assessment of surface righting reflex on Day 1
- Individual bodyweight and litter weight of live pups on days 1 and 4.
- Bodyweight gain from days 1 to 4

Postmortem examinations (parental animals):
- Terminal sacrifice, Males: Killed on day 43.
Terminal sacrifice, Females: Killed on day 5 post partum. Females without delivery were killed on or after Day 26 post coitum.

- Gross pathology: All rats received a full macroscopic examination with tissue collection.

- Organs Weights: Testes and epididymides were weighed at necropsy.

- Histopathology: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
Coagulating gland, epididymides, ovaries, mammary tissue (females only), pituitary, prostate, seminal vesicles,
testes, uterus/cervix, vagina

Postmortem examinations (offspring):
- Full external and internal gross pathology of all pups (terminal sacrifice and interim deaths)
Bodyweight and bodyweight change, food consumption for females during gestation and lactation, litter data, sex ratios, implantation losses and viability indices, offspring bodyweight and bodyweight change, offspring surface righting, adult absolute organ weights and organ to bodyweight ratios were assessed by linear regression analysis (for dose response relationships) followed by one way analysis of variance (ANOVA) incorporating Levne’s test for homogeneity of variance.
- Where variances were shown to be homogeneous pairwise comparisons were made using Dunnett’s test.
- Where Levene’s test showed unequal variances the data were analysed non- parametrically using Kruskal-Wallis ANOVA and Mann-Whitney “U’ test.

The non-parametric methods were also used to analyse implantation loss and offspring sex ratios and landmark developmental markers.
Reproductive indices:
- No. of pairs with successful mating
- Pre-coital interval (time, i.e. no. of days, elapsing between initial pairing and positive evidence of maiting)
- Mating index (No. of animals mated / No. of animals paired x 100)
- Pregnancy index (No. of pregnant females / No. of females mated x 100)
- Gestation length , i.e. no. of days including the days of positive evidence of mating, gestation and parturition start
- Parturition index (No. of females delivering live pups / No. of living pregnant females x 100)
- No. of corpora lutea / dam
- No. of implants / dam
- % Pre-implantation loss [(No. of corpora lutea – No. of implantation sites) x 100 / No. of corpora lutea]
- % Post-implantation loss [(No. of implantation sites – No. of live pups) x 100 / No. of implantation sites]
- For further reproductive parameters, see also the above section "Litter observations"
Offspring viability indices:
- Live birth index (No. of live pups on day 1 / No. of pups born on day 0 x 100)
- Viability index (No. of live pups on day 4 / No. of live pups on day 1 x 100)
- Post natal loss of pups (live births minus pups alive on day 4)
- For further parameters indicative of the viability of the offspring, see also the above section "Litter observations"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Description (incidence and severity):
recording of estrous only during the mating period
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
on testes and epididymides weight or on the degree of sperm presence in vaginal smears
Reproductive performance:
no effects observed

Details on results (P0)

- There were no unscheduled deaths in parental animals during the study.

Clinical Observations:
- Incidental episodes of increased salivation immediately after dosing in high dose animals were considered not indicative of systemic toxicity, but
may have been a reflection of unpleasant tasting test material formulation.
- Isolated episodes of noisy respiration, fur loss, scab formation or staining around the eyes or in the ano-genital region in various dose groups were considered to be incidental findings not representing an adverse treatment effect.

Water Consumption:
- Visual inspection of water bottles did not reveal any overt effects on water consumption.

Reproductive performance:
- Mating: No adverse effects, all animals from the control and treatment groups mated within 5 days of pairing.
- Fertility: No adverse effects, only 1 low dose and 1 high dose female did not achieve pregnancy (confirmed by absence of
corpora lutea and of implantation sites in these 2 females). All others gave birth to live litters.
- Gestation length: No adverse effects, gestation lasted 22 to 23.5 days in all pregnant females.
- Other parameters: No intergroup differences in numbers of corpora lutea or implantation sites or in implantation losses, litter size or
offspring viability attributable to treatment with the test material.

Gross Pathology:
- A yellow mass in the inguinal region in 1 mid dose male and small testes in 2 low dose males were the only findings
and were considered to be incidental and of no toxicological importance.

- Males:  No treatment-related abnormality was observed.
- Females:  No treatment-related abnormality was observed.
There were no histopathological indications why the non-pregnant high dose female did not achieve pregnancy.

Effect levels (P0)

Key result
Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: NOEL = highest dose tested.

Target system / organ toxicity (P0)

Key result
Critical effects observed:

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

No adverse effects or abnormalities attributable to treatment with the test material were observed.

Additional Data:
- No abnormality in sex ratio.
- No adverse effect on surface righting reflex.

Grossly Visible Abnormalities and External Abnormalities:
- At terminal kill in one mid dose pup a right testis, left uterine horn and ovary was present. The ovary was enlarged and dark.

In addition, there were 3 interim deaths in the high dose group and 1 in the vehicle control group, of which 2 high dose pups and the control pup were autolytic and the other high dose pup was without macroscopic abnormalities.

Effect levels (F1)

Key result
Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
other: no direct dosing of F1 animals
Basis for effect level:
other: NOEL = highest dose tested.

Target system / organ toxicity (F1)

Key result
Critical effects observed:

Overall reproductive toxicity

Key result
Reproductive effects observed:

Applicant's summary and conclusion

There were no relevant effects attributable to treatment with the test material. The no observed effect level (NOEL) for maternal and foetal toxicity is
1000 mg/kg bw/day.