Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
Not applicable
Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15 April 2013 to 09 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducetd according to international test guidelines and to GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: males were approximately 10 weeks old and the females were approximately 9 weeks old.
- Weight at study initiation: males had a mean body weight of 373 g (range: 324 g to 431 g) and the females had a mean body weight of 214 g (range: 196 g to 245 g).
- Fasting period before study: no
- Housing: the animals were individually housed, except during mating (males and females together during nights) and lactation (females with their litter), in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen for mating trial purposes and since it is preferable for pregnant animals.
Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material.
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet, batch Nos. 4898480 (SSNIFF Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): tap water (filtered with a 0.22 µm filter).
- Acclimation period: 8 days before the beginning of the treatment period. One supplementary animal per sex was acclimated to permit the selection and/or replacement of individuals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h.

IN-LIFE DATES: From: 16 April 2013 to 09 June 2013.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
(PEG 400)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as an emulsion in the vehicle. It was mixed with the required quantity of vehicle. No correction factor was applied for the dose calculations.
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw/day
- Lot/batch no. (if required): MKBG6036V
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 15 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually.
- Any other deviations from standard protocol: none
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of the test item in the dose formulations have been quantified by a validated Ion Chromatography (IC) with conductivity detection analytical method.
The validation of the analytical method was conducted in CiToxLAB France/Study No. 40028 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results are documented in the corresponding validation report.
Duration of treatment / exposure:
In the males:
- 2 weeks before mating (from study day 1 to 14)
- during the mating period (from study day 15 until study day 16 to 19),
- until sacrifice (at least 5 weeks in total) (from study day 17 to 20 until study day 36).

In the females:
- 2 weeks before mating (from study day 1 to 14),
- during the mating period (from study day 15 until study day 16 to 19),
- during gestation (from study day 16 to 19 until study day 36 to 40),
- during lactation until day 5 p.p. inclusive (from study day 37 to 41 until study day 42 to 46),
- until sacrifice for the non-pregnant female (until study day 42).

Day 1 corresponds to the first day of the treatment period.
Frequency of treatment:
Once daily, at approximately the same time (7 days/week)
Details on study schedule:
- Age at mating of the mated animals in the study: 12 weeks for males and 11 weeks for females.
Remarks:
Doses / Concentrations:
0, 100, 400 and 1000 mg/kg bw/day.
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals/sex/dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose-levels were set based on the results of a previous 2-week dose-range finding study performed in the same species and strain (CiToxLAB France/Study No. 40031 TSR). In this study, four groups of three males and three females received 0 (vehicle: PEG 400), 100, 400 or 1000 mg/kg/day daily for 2 weeks by gavage with a constant dose-volume of 5 mL/kg/day. There was no mortality and clinical signs consisting in piloerection, hunched posture, soft feces and ptyalism have been observed sporadically at 400 (in one male and all females) and 1000 (in all animals) mg/kg/day in the second week of treatment. There were no obvious effects on mean body weight, mean food consumption and mean organ weights. There were no adverse macroscopic findings at necropsy (distended intestinal track was present in 1/3 male from 100 mg/kg/day). Therefore, the same dose-levels were used for this main study.

- Rationale for animal assignment (if not random): during the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition. The animals were allocated to groups (by sex) using a computerized stratification procedure based on body weight, so that the average body weight of each group was similar.
Positive control:
Not used.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each animal was checked for mortality or signs of morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays. From arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs. Additionally, the females were observed for signs of difficult or prolonged parturition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 post-coitum (p.c.) and days 1 and 5 p.p..

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was measured once a week, over a 7 day period, from the first day of treatment until the start of the mating period. The quantity of food consumed by each female was measured once a week, over a 7 day period, from the first day of treatment until the start of the mating period, during pregnancy at the intervals days 0-7, 7 14 and 14-20 p.c. and during lactation for interval days 1 5 p.p.. During the mating period, food consumption was not measured for males or females.

Other: See section 7.5.1 (Repeated dose toxicity (Rep. dose tox. oral V1 CiToxLAB 2013)



Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage, each morning during the mating period, until the females were mated.
Sperm parameters (parental animals):
Parameters examined in P1 male parental generation: testis weight and epididymis weight.
Litter observations:
STANDARDISATION OF LITTERS
Not applicable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, live births, postnatal mortality, presence of gross anomalies, weight gain and physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: after the end of the mating period (at least 5 weeks of treatment in total). All animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.
- Maternal animals: on day 6 p.p.. All animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.
GROSS PATHOLOGY: Yes
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues, including the urinary bladder and the internal surface of the esophagus and stomach, and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
The numbers of corpora lutea and implantation sites were recorded for females sacrificed as scheduled on day 6 p.p. and one female (group 3) sacrificed on day 25 p.c. for no delivery.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table (See Table 7.5.1/1 section 7.5.1 (Repeated dose toxicity (Rep. dose tox. oral V1 CiToxLAB 2013)) from the first five sacrificed as scheduled males and the first five females sacrificed on day 6 p.p. of the control and high-dose groups (groups 1 and 4),
- forestomach, stomach, duodenum, jejunum and ileum from the first five sacrificed as scheduled males and the first five females sacrificed on Day 6 p.p. of the low- and intermediate-dose groups (groups 2 and 3) following the results obtained on those organs at macroscopy and/or microscopy in the high-dose group,
- reproductive organs from one female and one male (group 3) that did not conceive to investigate possible causes,
- all macroscopic lesions of all groups.

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed by an intraperitoneal injection of sodium pentobarbital on day 5 p.p..
- These animals were subjected to postmortem examinations (macroscopic examination)
Statistics:
The following statistical methods were used to analyze body weights, food consumption, reproduction and skeletal examination data:
• Means and standard deviations of various data were calculated.
• If the variables could be assumed to follow a normal distribution, the Dunnett t-test, based on a pooled variance estimate, was used for inter-group comparisons (i.e. single treatment groups against the control group).
• The Steel test (rank test) was applied when the data could not be assumed to follow a normal distribution.
• Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
- pre-implantation loss: Number of corpora lutea - Number of implantation sites/Number of corpora lutea x 100.

- post-implantation loss (manually calculated): Number of implantation sites - Number of live pups/Number of implantation sites x 100.

- mating index:Number of mated animals/Number of paired animals x 100.

- fertility index: Number of pregnant female partners/Number of mated pairs x 100.

- gestation index: Number of females with live born pups/Number of pregnant females x 100.



Offspring viability indices:
- live birth index: Number of live born pups/Number of delivered pups x 100.

- viability index on day 4 post-partum: Number of surviving pups on day 4 post-partum/Number of live born pups x 100.

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Observed clinical signs were considered to be related to the test item but of minor toxicological significance
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:
effects observed, treatment-related
Description (incidence and severity):
See details on results section
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths during the study. One female treated at 400 mg/kg/day was sacrificed on day 25 p.c. for no delivery (at necropsy, this female was not pregnant).
Ptyalism was noted in up to 6 animals per sex at 400 mg/kg/day and in all animals at 1000 mg/kg/day. This clinical sign was considered to be related to the test item (dose-relationship response and no control animals with this clinical sign) but of minor toxicological significance. There were no test item-related clinical signs at 100 mg/kg/day.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no test item-related effects on mean body weight and body weight gain.
Mean body weights were similar among groups as well as mean body weight gains in females over the pre-mating, gestation and lactation periods. Mean body weight gains in test item-treated males over the treatment period were slightly higher than in controls; however, there were no dose-relationship and no statistical level and they did not impact toxicologically the mean body weights. There were considered to be fortuitous.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no test item-related effects on mean mating and fertility data. All animals mated within comparable mean number of days. One female given 400 mg/kg/day was not pregnant which was considered to be incidental (not dose-related and isolated).There were no test item-related effects on mean delivery data. Mean duration of gestation was comparable between groups, as well as mean number of corpora lutea, implantations and pups delivered. Mean pre- and post-implantation loss of test item groups were not impacted.


ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test item-related mean organ weight differences.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The dose-related thickening of small intestine was considered to be related to test item administration. It was seen in one female at 400 mg/kg/day (duodenum) and in 5 females treated at 1000 mg/kg/day at duodenum level and in one additional female treated at 1000 mg/kg/day at both jejunum and ileum levels. This correlated to the hyperplasia of crypts seen microscopically (except for the ileum for which no microscopic correlate was seen).
The other macroscopic findings had no histological correlates or correlated with common histological findings in control rats, and were thus considered to be incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test item-related microscopic findings were seen in the forestomach, stomach, duodenum and jejunum of animals treated at 400 or 1000 mg/kg/day. Minimal to moderate squamous cell hyperplasia accompanied by hyperkeratosis was noted in the forestomach from males and females treated at 400 or 1000 mg/kg/day. In the stomach from one female treated at 1000 mg/kg/day, a slight atrophy together with glandular basophilia suggestive of regeneration was seen. This lesion was considered to be related to test item administration, while the minimal atrophy seen in one female treated at 100 mg/kg/day was considered not to be related to test item treatment since it was minimal and isolated, since no lesions were seen at 400 mg/kg/day and since there was no evidence of regeneration. In the duodenum from males and females treated at 400 or 1000 mg/kg/day, there was minimal to slight hyperplasia of crypt, accompanied by Brunner’s gland hyperplasia in males treated at 1000 mg/kg/day only. In the jejunum from one female treated at 1000 mg/kg/day, there was also minimal hyperplasia of crypt. The hyperplasia of crypt correlated to the thickening of duodenum/ileum seen macroscopically. In view of the low to moderate magnitude of these changes, of the absence of associated degenerative changes and of the absence of body weight changes, these findings were considered not to be adverse and suggested local irritant properties of the compound.
There was minimal infiltrate of eosinophils in the rectum from two females treated at 1000 mg/kg/day and in the stomach from one male treated at 400 mg/kg/day, while not seen in controls. The relationship of this finding to test item-treatment was considered to be unlikely in view of the low incidence and severity of this change. It is noteworthy that the urinary bladder from the animals treated at 1000mg/kg/day was within normal limits.
The reproductive organs from the female treated at 400 mg/kg/day and sacrificed on day 25 p.c. because of no delivery (non-pregnant) and fromthe male, mated with female did not show any abnormalities.


OTHER FINDINGS (PARENTAL ANIMALS): See section 7.5.1 (Repeated dose toxicity (Rep. dose tox. oral V1 CiToxLAB 2013)
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no effects in the mating, fertility and gestation rates or in pre- and post-implantation losses.
Remarks on result:
other: Generation: P/Reproduction (mating and fertility)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
There were no test item-related effects on pup mortality. At 100 mg/kg/day, 7 pups vs. 3 in controls were found dead; however, this was due to one litter(B20812) with 6 found dead pups (autolysed or with no milk in the stomach) on day 1 p.p.. This was thus not considered to be test item-related as it was an isolated event and no effect was seen in the higher doses groups.

CLINICAL SIGNS (OFFSPRING)
There were no test item-related effects on clinical signs. Incidences in test item-treated groups were comparable to or lower than those of controls.

BODY WEIGHT (OFFSPRING)
There were no test item-related effects on pup mean body weight and pup mean body weight gain as there were no statistically and toxicologically significant differences between groups and no dose-relationship.

GROSS PATHOLOGY (OFFSPRING)
There were no test item-related macroscopic findings at pup necropsy. The only statistically significant difference was observed at 100 mg/kg/day on the total macroscopic observations in found dead pups and was not considered to be related to the test item treatment. This was due to the high mortality rate in litter B20812 at this dose-level, not ascribed to the test item treatment.

OTHER FINDINGS (OFFSPRING): Pup sex ratio:
There were no test item-related effects on the percentage of male pups at birth which was comparable among groups and close to 50%. Moreover, there were no statistically significant differences from the control group.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no effects in pup, mortality, clinicals signs, sex ratio, mean body weight or macroscopic post-mortem findings
Reproductive effects observed:
not specified

No remarks.

Conclusions:
Under the test conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 1000 mg/kg/day based on ptyalism (minor toxicological significance), minor blood biochemistry findings observed in males and non-adverse pathology findings noted at this dose-level. No reprotoxic effects were observed in all treated animals. Therefore, the NOEL for reproduction, development and fertility was considered to be 1000 mg/kgbw /day (the highest tested dose).
Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (CiToxLAB, 2013) was conducted with Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate according to the OECD test guideline No. 422 and in compliance with GLP.

Sprague-Dawley rats (10/sex/dose level) received the test item daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until day 5 p.p.. The test item was administered at dose levels of 100, 400 and 1000 mg/kg/day as an emulsion in the vehicle (Polyethylene Glycol 400, PEG 400). Another group of ten males and ten females received the vehicle, alone, under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg bw/day was used.

The animals were checked daily for clinical signs, mortality, and detailed clinical observations were conducted weekly. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation.

The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 5 p.p.. The total litter sizes and numbers of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behaviour, external abnormalities and weighed on Days 1 and 5 p.p..

A Functional Observation Battery (FOB), including motor activity, and laboratory investigations (hematology, blood biochemistry and urinalysis) were carried out on five males and five females from each group at the end of the study.

The males were sacrificed after at least 5 weeks of treatment and the females on day 6p.p.. Final body weights and selected organs weights(adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus and urinary bladder) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs, the urinary bladder and the internal surface of the esophagus and stomach. A microscopic examination was performed on selected organs (including the urinary bladder) from five males and five females in the control and high-dose groups, on forestomach, stomach, duodenum, jejunum and ileum from five males and five females of the low- and intermediate-dose groups, and on all macroscopic lesions. The pups were sacrificed on day 5p.p.and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.

There were no unscheduled test item-related deaths.Ptyalism was the only recorded clinical sign,noted in up to 6 animals per sex at 400 mg/kg/day and in all animals at 1000 mg/kg/day.This finding was considered to be related to the test item but of minor toxicological importance.There were no effects on body weight, food consumption, functional observation battery tests or motor activity data in any group and sex. At laboratory investigations, no relevant changes were noted. Mating, fertility and delivery data were unaffected by the test item treatment. No effects on pups were observed up to Day 5p.p.(viability, clinical signs, sex ratio, macroscopic post‑mortem findings). There were no test item-related mean organ weights differences. The dose-related thickening of duodenum, jejunum and/or ileum seen in females treated at 400 or 1000 mg/kg/day was considered to be related to test item administration. Test item-related non-adverse microscopic findings were seen in the forestomach (squamous cell hyperplasia; hyperkeratosis); stomach (glandular atrophy), duodenum (crypt and Brunner’s gland hyperplasia) and jejunum (crypt hyperplasia) of animals treated at 400 or 1000 mg/kg/day, and suggested local irritant properties of the test item.

Based on the experimental conditions of this study:

- The No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 1000 mg/kg/day based on ptyalism (minor toxicological significance), minor blood biochemistry findings (decrease in mean total bilirubin and protein levels in males) and non-adverse pathology findings (microscopic findings in the forestomach, stomach, duodenum and jejunum suggesting local irritant properties of the compound) noted at this dose-level,

- The No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day as there were no effects in the mating, fertility and gestation rates or in pre- and post-implantation losses,

- The NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day as there were no effects in pup mortality, clinical signs, sex ratio, mean body weight or macroscopic post-mortem findings.

This study is considered as acceptable as it satisfies the main criteria of OECD guideline No.422.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD guideline 422 reprotoxicity screening study (Kr. 1).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

One study of reliability 1 according to Klimisch cotation critera, is available (CiToxLAB, 2013) and was selected as a key study.

In this study (OECD 422), Sprague-Dawley rats (10/sex/dose level) received the test item daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until day 5 p.p.. The test item was administered at dose levels of 100, 400 and 1000 mg/kg/day as an emulsion in the vehicle (Polyethylene Glycol 400, PEG 400). Another group of ten males and ten females received the vehicle, alone, under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg bw/day was used.

The animals were checked daily for clinical signs, mortality, and detailed clinical observations were conducted weekly. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation.

The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 5 p.p.. The total litter sizes and numbers of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behaviour, external abnormalities and weighed on Days 1 and 5 p.p..

A Functional Observation Battery (FOB), including motor activity, and laboratory investigations (hematology, blood biochemistry and urinalysis) were carried out on five males and five females from each group at the end of the study.

The males were sacrificed after at least 5 weeks of treatment and the females on day 6 p.p.. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus and urinary bladder) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs, the urinary bladder and the internal surface of the esophagus and stomach. A microscopic examination was performed on selected organs (including the urinary bladder) from five males and five females in the control and high-dose groups, on forestomach, stomach, duodenum, jejunum and ileum from five males and five females of the low- and intermediate-dose groups, and on all macroscopic lesions. The pups were sacrificed on day 5 p.p.and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.

There were no unscheduled test item-related deaths. Ptyalism was the only recorded clinical sign,noted in up to 6 animals per sex at 400mg/kg/day and in all animals at 1000 mg/kg/day.This finding was considered to be related to the test item but of minor toxicological importance.There were no effects on body weight, food consumption, functional observation battery tests or motor activity data in any group and sex. At laboratory investigations, no relevant changes were noted. Mating, fertility and delivery data were unaffected by the test item treatment. No effects on pups were observed up to Day 5p.p.(viability, clinical signs, sex ratio, macroscopic post‑mortem findings). There were no test item-related mean organ weights differences. The dose-related thickening of duodenum, jejunum and/or ileum seen in females treated at 400 or 1000 mg/kg/day was considered to be related to test item administration. Test item-related non-adverse microscopic findings were seen in the forestomach (squamous cell hyperplasia; hyperkeratosis); stomach (glandular atrophy), duodenum (crypt and Brunner’s gland hyperplasia) and jejunum (crypt hyperplasia) of animals treated at 400 or 1000 mg/kg/day, and suggested local irritant properties of the test item.

Based on the experimental conditions of this study:

- The No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 1000 mg/kg/day based on ptyalism (minor toxicological significance), minor blood biochemistry findings (decrease in mean total bilirubin and protein levels in males) and non-adverse pathology findings (microscopic findings in the forestomach, stomach, duodenum and jejunum suggesting local irritant properties of the compound) noted at this dose-level,

- The No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day as there were no effects in the mating, fertility and gestation rates or in pre- and post-implantation losses,

- The NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day as there were no effects in pup mortality, clinical signs, sex ratio, mean body weight or macroscopic post-mortem findings.


Short description of key information:
OECD 422 Guideline study:
the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 1000 mg/kg/day based on ptyalism (minor toxicological significance), minor blood biochemistry findings observed only in males and non-adverse pathology findings noted at this dose-level. No reprotoxic effects were observed in all treated animals. Therefore, the NOEL for reproduction, development and fertility was considered to be 1000 mg/kgbw /day (the highest tested dose).

Justification for selection of Effect on fertility via oral route:
Only one study is available.

Effects on developmental toxicity

Description of key information

One study of reliability 1 according to Klimisch cotation critera, is available (CiToxLAB, 2017) and was selected as a key study.

In this study (OECD 414), three groups of 24 time-mated female Sprague-Dawley rats received the test item, Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (batch No. 1629110), by the oral route (gavage), at a dose-level of 100, 400 or 1000 mg/kg/day, once daily from Day 6 to Day 20 p.c. inclusive. Another group of 24 time-mated rats received the vehicle only, Polyethylene Glycol 400 (PEG 400), under the same experimental conditions, and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. The test item concentrations were determined in the dose formulations. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 21 p.c., the females were sacrificed and a macroscopic post-mortem examination was performed. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities (including cartilage).

At 1000 mg/kg/day, one pregnant female was found dead and seven pregnant females were prematurely sacrificed. In absence of microscopic examination, the cause of death cannot be formally established but clinical signs and macroscopic observations are suggesting that corrosivity is responsible. In addition to the mortalities the following clinical signs have been observed : ptyalism, round back and/or piloerection from 100 mg/kg, emaciated appearance in isolated animals from 400 mg/kg/day and loud and/or abdominal breathing defined as endpoints, pallor of extremities and/or soft feces at 1000 mg/kg/day. While terminal body weight and food consumption were not affected anymore at the end of the study, there was a correlated body weight gain and food consumption reduction on days 9 -12 p.c. and days 15 -18 p.c. in the 1000 mg/kg/day group. These reductions were mainly related to effects to animals which have sacificaed afterwards as well as to two surviving females J21524 and J21526. These two females macroscopic examinations showed observations consistent with corrosive effects (whitish colored stomach mucosa, dilatation of intestines, associated with reduced thymus or enlargement of one adrenal gland)

At study termination on Day 21 p.c.,there were 24, 23, 24 and 16 females with live fetuses in the groups given 0, 100, 400 and 1000 mg/kg/day, respectively. There were no test item treatment-related effects for hysterectomy data.

There was no toxicologically important effect on mean fetal body weight and the sex ratio was not affected by the test item treatment at any dose-level.

There were no test item-related variations or malformations at external examination.

At soft tissues examination there were higher litter and fetal incidences of dilated ureter at all dose-levels, however this variation was considered as non adverse. One malformed fetus had marked dilatation of the ureter but this as the incidence of this malformation was isolated and within the Historical Control Data, this was considered to be of no toxicological importance.

At 1000 mg/kg/day and when compared with controls, there were higher litter and fetal incidences of fetuses with delayed ossification: hyoid (unossified), caudal vertebra(e)(unossified centrum and/or incomplete ossification of arch and/or centrum), 6thsternebra (unossified or incomplete ossification) and 1st metatarsal bone (unossified). This was considered to be associated with the test item treatment but of minor importance (see § Cartilage), although the incidences were below the maximum values in the Historical Control Data in some cases. These findings were consistent with the slightly lower mean fetal body weight recorded at this dose-level and were also mainly due to the contribution of fetuses from females J21524 and J21526 who presented signs of maternal toxicity.

In the control group, one litter contained one malformed fetus: J21449-05 with split interparietal bone. In the 1000 mg/kg/day group, one litter contained one malformed fetus (fused sternebrae) and another one contained three malformed fetuses[absent lumbar vertebra(e)], for which a test item treatment-related effect cannot be excluded.

The fused sternebrae malformation observed is within Historical Control and can be considered as incidental.

The absence of lumbar vertrebrae has been observed on three fetuses which is slightly higher than in Historical Control Data but surprisingly in only one litter which is well within Historical Control Data making this malformation restricted to one animal. Therefore, this observation taken together with the context of strong maternal toxicity observed during this study, makes it unlikely that this specific malformation is linked to a direct effect of the tested substance on the fetuses even if it cannot be completely excluded.


Conclusion 

The test item, Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (batch No. 1629110) was administered by gavage, once daily, from Days 6 to 20p.c.,inclusive, to pregnant Sprague-Dawley rats at the dose-levels of 100, 400 and 1000 mg/kg/day.The control group received the vehicle, Polyethylene Glycol 400 (PEG 400), under the same experimental conditions. 

On the basis of the results obtained in this study:

.          the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 400 mg/kg/day (based on clinical signs, food consumption, body weight gain and mortality at 1000 mg/kg/day),

.          the NOAEL for embryo-fetal development was considered to be 400 mg/kg/day (based on fetal dysmorphogenesis at 1000 mg/kg/day) in a context of severe maternal toxicity.


Short description of key information:
In the OECD 422 Guideline study, no effects were observed on the pups up to 1000 mg/kg/day while only minor toxicological effects were seen for parental toxicity.

In the OECD 414 Guideline study, stronger parental toxicity was observed at the 1000 mg/kg/day with observations consistent with corrosive effect probably linked to a difference between the test substance batches. In this context of severe maternal toxicity, some non-adverse variations and a few malformations were observed, the latter restricted to a single litter.


Justification for selection of Effect on Developement via oral route:
Only one OECD 414 study is available.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2016-11-02
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: at the beginning of the treatment period, the animals were 10-11 weeks old
- Weight at study initiation: mean body weight of 272 g (range: 227 g to 309 g)
- Fasting period before study: no
- Housing: The animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen in order to not jeopardize gestations.
Each cage contained nylabone and rat hut for the environmental enrichment of the animals.
The cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 799891 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: the animals were acclimated to the study conditions for a period of 4 or 5 days before the beginning of the treatment period (arrival of the females on Day 1 or 2 p.c.).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30% - 70%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From 2016-11-17 to 2016-12-15
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as an emulsion in the vehicle. The required quantities were mixed progressively with the vehicle in order to obtain the desired concentration. After addition of the whole quantity of vehicle, the dose formulations were kept under magnetic stirring for at least 30 minutes to ensure effective solubilization of the test item. Based on test item dose formulation stability CiToxLAB France/Study No. 40029 AHS, the test item dose formulations were prepared on each day of treatment, stored and delivered at room temperature and protected from light. Control dose formulations were stored and delivered each day at room temperature.


VEHICLE
- Concentration in vehicle: 0, 20, 80 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/ kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Principle of the analytical method
The analytical method was developed at CiToxLAB France. It consisted of sampling 1 mL of dose
formulation and diluting it appropriately with diluent to reach the nominal concentration of injection
(0.1 mg/mL). The diluted samples were analyzed by Ion Chromatography (IC) with conductivity detection,
bracketed by Quality Control samples and quantified using a calibration curve. The quantification was
based on the analysis of one component of the test item considered as a marker. The peak area of this
marker was used for quantification.

Solvent and reagent
Milli-Q water (CiToxLAB France)
Sodium Hydroxide S5881-500g (Sigma-Aldrich)
Sulfuric acid 320501-1L (Sigma-Aldrich)
Diluent: 0.01M sodium hydroxide.

Equipment
High Performance Liquid Chromatography Ionic system with conductivity detection (ICS5000) (Thermo)
Micro-balance; Balance (Mettler-Toledo)
Automatic pipettes (Biohit)
equipments for agitating (i.e. magnetic stirring, vortex…),
class A volumetric flasks.

Software
Empower 2 (Waters).

Samples preparation
For the determination of nominal concentrations, the dilution volumes used were calculated by measuring the weight of each sampling and assuming the density of the dose formulation sampled (d = 1.13).
Dilution factor = (V2 x V1) / (v1 x v2)
With:
V1 = total volume for the first dilution
V2 = total volume for the second dilution
v1 = volume taken for each sample (with v1 = weight of sample / density of the formulation)
v2 = volume taken for the second dilution

Composition of the analytical sequence
Analytical sequences are composed of at least:
. a blank sample (diluent) was checked for the absence of chromatographic interferences, . vehicle sample (when requested),
. a LOQ solution
. a calibration curve using five standard solutions at increasing concentrations within the range defined during validation
. at least two Quality Control (QC) samples at one concentration level prepared using independent standard solutions (different to those used for the calibration curve)
. study samples prepared from aliquots of the dose formulations bracketed by the QC samples.

Analytical note: for the determination of content of administered dose formulations analyzed in Week 3 (07 December 2016), the LOQ solution prepared at 2 μg/mL was not detected. The LOQ solution provided direct estimations of potential contamination in the group control. No interfering peaks was detected in the group control on 07 December 2016, thus this deviation to internal SOP has no impact on the results.

Quantification
The quantification was based on the analysis of one component of the test item, considered as a marker. Peak areas of this marker were used for the quantification.
Peak areas were determined for standard solutions ranging from 0.05 to 0.15 mg/mL of Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (five levels). A calibration curve was obtained by linear regression analysis of peak areas against concentrations and the regression analysis of the calibration data gave an equation as follows:
Y = a X + b
where:
Y = peak area of Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (μV.s)
X = concentration of Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (mg/mL)
a = slope value
b = intercept

Acceptance criteria of an analytical sequence
Acceptance criteria are defined in CiToxLAB France Standard Operating Procedures (SOPs).
The acceptance criteria included:
. coefficient of determination: r2 ≥ 0.98,
. deviation calculated between the theoretical and measured concentrations (%) for calibration points
and Quality Controls: between -7% and +7%.
%DEV = (Cm-Ct)Ct x 100
With: Cm: measured concentration
Ct: theoretical concentration

Dose formulation analysis
Assay
Diluted samples of dose formulations were analyzed in single by Ion Chromatography (IC) with conductivity detection.
The test item peak area was determined for each sample and the corresponding concentration was calculated using the equation obtained from the calibration curve.
All the results are expressed as mg/mL of Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate.

Acceptance criterion
Deviation calculated between measured concentration and theoretical concentration should be within
± 15%.
Details on mating procedure:
the females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as Day 0 p.c.
Duration of treatment / exposure:
The dose formulations were administered daily from Day 6 to Day 20 p.c., inclusive.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
400 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:
24 animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement, on the basis of the results of a previous OECD 422 study performed in the same species and strain (CiToxLAB France/Study No. 40032 RSR), in which four groups of 10 males and 10 females received 0 (PEG 400), 100, 400 or 1000 mg/kg/day, daily by gavage, under a constant dosage volume of 5 mL/kg/day.
There were no test item-related deaths, and clinical signs consisted of ptyalism that was noted in up to six animals per sex at 400 mg/kg/day and in all animals at 1000 mg/kg/day. There were no test item treatment-related effects on mean body weight, mean food consumption, functional observation battery, mean hematological and urinalysis parameters or mean organ weights. There were no test item treatment-related effects on mating, fertility and delivery data, or on mortality, clinical signs, sex ratio, mean body weight or macroscopic post-mortem findings in pups.
There were minor blood biochemistry findings (decrease in mean total bilirubin and protein levels) in males given 1000 mg/kg/day. There were non-adverse pathology findings (thickening of duodenum, jejunum and/or ileum in females from 400 mg/kg/day and microscopic findings in the forestomach, stomach, duodenum and jejunum) suggesting local irritant properties of the compound in males and females from 400 mg/kg/day.
Therefore, the same dose-levels were used for this study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c., and prior to premature euthanasia.

FOOD CONSUMPTION: Yes
-The quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: the principal thoracic and abdominal organs
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- number of corpora lutea
- number and distribution of dead and live fetuses
- number and distribution of early and late resorptions
- number and distribution of uterine scars
- number and distribution of implantation sites.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes
Statistics:
Data were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by Fisher exact probability test (proportions).
Historical control data:
Yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item treatment-related clinical signs consisted of:
- round back and/or piloerection observed in the second or third week of treatment in females at all dose-levels,
- ptyalism, usually observed from the second week of treatment in females given 100 or 400 mg/kg/day or from the first week of treatment in females given 1000 mg/kg/day,
- emaciated appearance noted from the second week of treatment in females given 400 or 1000 mg/kg/day,
- loud and/or abdominal breathing, pallor of extremities and/or soft feces observed from the second or third week of treatment in females given 1000 mg/kg/day.
Breathing difficulties were considered to be endpoints and therefore to have exceeded the Maximal Tolerated Dose.

Reflux at dosing (noted on one occasion in 1/24 females given 100 mg/kg/day) resulted from the gavage procedure. Reddish vaginal discharge observed in 1/24 control females and 2/24 females given 400 mg/kg/day was considered to be unrelated to the test item treatment, as it was reported on a limited number of occasions and without any dose relationship.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no test item treatment-related deaths in control animals or the 100 and 400 mg/kg/day treated groups.
At 1000 mg/kg/day, one pregnant female was found dead (female J21511) on Day 18 p.c., and on Day 13, 18, 19 or 20 p.c., seven pregnant females were prematurely sacrificed (females J21509, J21514, J21515, J21516, J21520, J21521 and J21522). Prior to death, these females showed signs of poor clinical condition (i.e. piloerection, round back, pallor of extremities, emaciated appearance, hypotonia, hypoactivity, abdominal and/or loud breathing, dyspnea, chromodacryorrhea, chromorhinorrhea, half-closed eyes, soft feces, and/or soiled head or urogenital area). Ptyalism was also noted in all females. Moderate to severe body weight loss was noted in all females (between -23 to -83 g) over the last intervals, together with reduced food consumption in 4/8 females (3 to 17 g/animal/day).
At macroscopic post-mortem examination, the adrenal glands were enlarged (2/8 females), the spleen was reduced in size (5/8 females), the stomach was dilated (7/8 females) and whitish colored content was present (1 female), and there was whitish colored mucosa (4 females), thickening of mucosa (2 females) and/or whitish or reddish colored foci (2 females). The intestines were dilated (8/8 females) and/or the lungs were dilated or with whitish liquid content in the thoracic cavity (1/8 females).
These unscheduled deaths were considered to be test item- related. Macroscopic observations are consistent with the hypothesis that the deaths are linked to corrosive properties of the test item. However, in absence of microscopic examination it cannot be confirmed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day and when compared with controls, slightly to markedly lower mean body weight gain was noted on Days 9-12 p.c. (+18 g vs. +24 g in controls) and on Days 15-18 p.c. (+20 g vs. +42 g in controls, p < 0.001). These variations were mainly due to the contribution of the body weight loss recorded in moribund females before their death and in surviving females J21524 and J21526. These findings were considered to be adverse although the terminal mean body weight on Day 21 p.c. was not affected.
Effects on mean body weight changes correlated with reduced food consumption between Days 15 and 18 p.c.

At 100 or 400 mg/kg/day, there were no effects on mean body weight or mean body weight change.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day and, when compared with controls, lower mean food consumption (-10%, p < 0.05) was recorded between Days 15 and 18 p.c., with a return to control values thereafter. This variation was mainly due to the contribution of the body weight loss recorded in moribund females before their death and in surviving females J21524 and J21526. This change was considered to be adverse. This difference correlated with the lower mean body weight gain also observed at 1000 mg/kg/day.

At 100 or 400 mg/kg/day, there were no effects of treatment with the test item.
Water consumption and compound intake (if drinking water study):
not examined
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):
At 400 and 1000 mg/kg/day, 1/24 (J21501) and 1/16 females (J21526), respectively, showed whitish colored stomach mucosa. Dilatation of intestines, associated with reduced thymus and enlargement of one adrenal gland, was also noted in another female (J21524) given the high-dose level. Since these findings were also observed in decedent females, they were considered to be test item treatment-related.
Like for the decedent females, these observations are consistent with the hypothesis that the deaths are linked to corrosive properties of the test item. However, in absence of microscopic examination it cannot be confirmed.
Other macroscopic findings (i.e. renal pelvis dilatation in 1/24 females given 400 mg/kg/day and enlargement of iliac lymph nodes in 1/16 females given 1000 mg/kg/day) were of isolated occurrence and/or not dose-related. They were, therefore, considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
In the 1000 mg/kg/day group, one dead fetus was observed. As the incidence of this finding was isolated and within the range of the Historical Control Data, it was considered to be incidental.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
See chapter general toxicity (Maternal animals)
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex-ratio (percentage of male fetuses) was not affected by the test item treatment at any dose-level.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
In the 1000 mg/kg/day group, when compared with controls and the HCD, mean fetal body weight was slightly lower ( 5%, p < 0.05). This variation was mainly due to the contribution of females J21524 and J21526 (net weight change:4.46 g and 4.30 g, respectively).As this test item treatment-related difference was minimal and did not correlate with a lower mean gravid uterus weight, it was considered to be of minor toxicological importance.
There were no effects on mean fetal body weight in the 100 and 400 mg/kg/day groups.

Litter size was not affected.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external variations or malformations at external fetal examination.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 400 and 1000 mg/kg/day and when compared with controls and Historical Control Data, there were higher litter and fetal incidences of fetuses with unossified hyoid for which cartilage was present (statistically significant at 1000 mg/kg/day).

At 1000 mg/kg/day and when compared with controls, there was an increase in delayed ossification of the caudal vertebrae (unossified centrum and/or incomplete ossification of arch and/or centrum), sternebrae (unossified or incomplete ossification 6th sternebra(e)] and metatarsal bone (unossified 1st metatarsal bone). This was considered to be associated with the test item treatment but of minor importance (see § Cartilage), although the incidences were below the maximum values in the Historical Control Data in some cases.
These findings were consistent with the slightly lower mean fetal body weight recorded at this dose-level and were also mainly due to the contribution of fetuses from females J21524 and J21526.

Other skeletal variations, [i.e. incomplete ossification of cervical vertebra(e) centrum observed in the 100 mg/kg/day group or dumbbell ossification of thoracic vertebra(e) centrum in the 400 mg/kg/day group] were considered to be unrelated to the test item treatment as they were not dose-related.

In the 1000 mg/kg/day group, two litters contained one or three malformed fetuses without correlate with any signs of maternal toxicity with the exception of loud breathing recorded in female J21503 from Day 18 to Day 20 p.c.

In the 100 and 400 mg/kg/day group, no malformed fetuses were observed.

In the control group, one litter contained one malformed fetus with split interparietal bone.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, there was a tendency towards increased fetal and litter incidences of dilated ureter at all dose-levels (from 6.7% to 7.6% and from 30.4% to 37.5%, fetal and litter incidences, respectively). Taking into account that incidences (fetal and litter) of this non-adverse finding were higher at the high-dose level than the maximum incidence in the Historical Control Data, it was considered to be test item treatment-related.

Other soft tissue variations (i.e. dilated renal pelvis, short or absent innominate artery, paleness of spleen, and/or reddish foci on thymus) observed with a similar or minimal incidence in control and/or test item treated groups were considered to be unrelated to the test item treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: See 'remarks'
Remarks on result:
other: see 'remarks'
Remarks:
in a context of severe maternal toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Pregnancy status

Dose-level (mg/kg/day)

        0

  100

       400

   1000

Number of females

      24

    24

         24

       24

Non-pregnant females

        0

      1

          0

        0

Found dead or prematurely sacrificed females

        0

      0

          0

        8

Females with total resorption

        0

      0

          0

        0

Females with live fetuses at term

      24

    23

         24

       16

 

Clinical signs

Test item treatment-related clinical signs (surviving females) are summarized as follows:

Dose-level (mg/kg/day)

0

100

400

1000

Round back

 

1
(for 2 days)

1
(for 3 days)

3
(
from 1 to 4 days duration)

Piloerection

 

1
(for 2 days)

2
(
for 1 or 6 days)

5
(
from 2 to 6 days duration)

Ptyalism

 

3
(from 1 to 5 days duration)

13
(from 3 to 10 days duration)

16
(from 3 to 15 days duration)

Reflux at dosing

 

1
(Day 18 p.c.)

 

 

Emaciated appearance

 

 

1a
(
from Day 13 to 20 p.c.)

1b
(
from Day 12 to 15 p.c.)

Loud breathing

 

 

 

2
(for 1 or 3 days)

Abdominal breathing

 

 

 

3
(from 1 to 5 days duration)

Pallor of extremities

 

 

 

1
(for 1 day)

Soft feces

 

 

 

1
(for 3 days)

Reddish vaginal discharge

1
(for 3 days)

 

2
(for 3 or 6 days)

 

Number of affected animals

1/24

5/24

15/24

16/16

( )  : in brackets: Daysp.c.of occurrences or length of the period.

a    : female J21494: +133 g between Days 12 and 21p.c.

b    : female J21507: - 29 g between Days 9 and 12p.c.

Body weight and body weight change

Dose-level (mg/kg/day)

Body weight (g)

0

100

400

1000

Day 6p.c.

271

271

271

275

-

(0)

(0)

(+1)

Day 9p.c.

287

289

289

290

-

(+1)

(+1)

(+1)

Day 12p.c.

311

313

310

309

-

(+1)

(0)

(-1)

Day 15p.c.

331

332

331

334

-

(0)

(0)

(+1)

Day 18p.c.

373

372

375

354

-

(0)

(+1)

(-5)

Day 21p.c.

433

429

430

422

-

(-1)

(-1)

(-3)

 Body weight change (g)

Days 9 - 12p.c.

+24

+24

+21

+18

Days 12 - 15p.c.

+20

+19

+21

+23

Days 15 - 18p.c.

+42

+40

+44

+20#

Days 18 - 21p.c.

+60

+57

+55

+54

Days 6 - 21p.c.

+162

+158

+158

+146

 

-

(-2)

(-2)

(-10)

Statistical significance;#: p < 0.001.

p.c.        :post-coitum.

-    : not applicable.

( )  : in brackets, percentage differencevs.controls.

 

Food consumption 

Dose-level (mg/kg/day)

0

100

400

1000

Days 6 - 9p.c.

26

-

27

(+4)

26

(0)

25

(-4)

Days 9 - 12p.c.

27

-

29

(+7)

27

(0)

27

(0)

Days 12 - 15p.c.

29

-

29

(0)

29

(0)

30

(+3)

Days 15 - 18p.c.

31
-

31
(0)

32
(+3)

28*
(-10)

Days 18 - 21p.c.

32

-

31

(-3)

31

(-3)

29

(-9)

Statistical significance;*: p < 0.05.

p.c.  :post-coitum.

-       : not applicable.

( )     : in brackets, percentage differencevs. controls.

 

Maternal necropsy findings

Dose-level (mg/kg/day)

0

   100

     400

  1000

Stomach: whitish colored mucosa

0

      0

        1

       1

Intestines: dilated

0

      0

        0

       1

Adrenal glands: enlarged

0

      0

        0

       1

Thymus: reduced in size

0

      0

        0

       1

Number of affected animals

0/24

0/24

1/24

2/16

 

Mean carcass, net change and gravid uterus weights

Dose-level (mg/kg/day)

0

100

400

1000

Gravid uterus weight

116

-

115

(-1)

116

(0)

112

(-3)

Carcass weight

317

-

314

(-1)

314

(-1)

310

(-2)

Net body weight change from Day 6.p.c.

+45

+43

+43

+35

( )     : in brackets, percentage differencevs.controls.

p.c.  :post-coitum.

(a)    : weights are rounded values.

-       : not applicable.

 

Hysterectomy data

Dose-level (mg/kg/day)

      0

  100

    400

1000

HCD

Number of pregnant females
at hysterectomy

     24

    23

     24

   16

      407

Number of females with live fetuses
at termination

     24

    23

     24

   16

      388

Number of females with total resorption

      0

      0

       0

     0

          0

Mean number ofcorpora lutea

     16.7

    17.2

     17.5

   18.0

[13.8; 16.0]

Mean number of implantation sites

     15.2

    15.4

     15.4

   15.6

[12.5; 14.5]

Mean pre-implantation loss (%)

      8.9

      9.3

     11.1

   11.5

[6.2; 14.0]

Mean number of live fetuses

     14.8

    14.8

     14.9

   15.3

[11.6; 13.8]

Dead fetuses (mean %)

      0.0

      0.0

       0.0

     0.40

[0.00; 0.50]

Mean number of implantation scars

      0.0

      0.0

       0.0

     0.0

/

Mean number of early resorptions

      0.3

      0.5

       0.5

     0.3

/

Mean number of late resorptions

      0.1

      0.1

       0.1

     0.1

/

Mean post-implantation loss (%)

      2.2

      3.8

       3.6

     2.3

[3.5; 11.1]

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016), [min.; max.].

/       : not reported in HCD.

 

Fetal body weight and sex

Dose-level (mg/kg/day)

0

100

400

1000

HCD

Mean fetal body weight (g)

5.74
-

5.73
(0)

5.77
(+1)

5.44*
(-5)

[5.5; 5.9]

Mean fetal body weight
of males (g)

5.91
-

5.88
(-1)

5.89
(0)

5.58*
(-6)

[5.7; 6.1]

Mean fetal body weight
of females (g)

5.57
-

5.58
(0)

5.63
(+1)

5.27*
(-5)

[5.4; 5.7]

Mean percentage
of male fetuses (%)

48.9

51.6

48.7

51.2

[44.0; 55.4]

( )     : in brackets, percentage differencevs.controls.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016), [min.; max.].

Statistically significant;*: p < 0.05.

-       : not applicable.

 

Fetal external variations

Dose-level (mg/kg/day)

       0

   100

   400

 1000

HCD

Dams with live fetuses

     24

     23

     24

     16

        388

Number of live fetuses

   356

   340

   357

   244

      5000

Litters affected, n (%)

0 (0)

0 (0)

0 (0)

0 (0)

6 (1.5)(b)

Fetuses affected, n (%)

0 (0)

0 (0)

0 (0)

0 (0)

7 (0.1)(b)

F      : fetal incidence.

L      : litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

 (b)    : mean incidence.

 

 

 

Fetal soft tissue variations

Dose-level (mg/kg/day)

        0

    100

    400

   1000

HCD

Dams with live fetuses

      24

      23

      24

      16

       387

Number of live fetuses

    171

    165

    175

    118

     2404

Litters affected, n (%)

6 (25.0)

9 (39.1)

7 (29.2)

8 (50.0)

86 (22.2)(b)

Fetuses affected, n (%)

11 (6.4)

15 (9.1)

16 (9.1)

12 (10.2)

119 (5.0)(b)

Dilated renal pelvis, F (L)

4.7 (12.5)

6.7 (34.8)

3.4 (16.7)

2.5 (18.8)

9.5 (28.6)(a)

Short innominate artery, F (L)

0 (0)

0 (0)

0 (0)

0.8 (6.3)

3.7 (22.7)(a)

Absent innominate artery, F (L)

1.2 (8.3)

1.2 (8.7)

1.1 (4.2)

0 (0)

5.1 (25.0)(a)

Dilated ureter, F (L)

4.7 (16.7)

6.7 (30.4)

6.9 (20.8)

7.6 (37.5)

7.1 (28.0)(a)

Spleen: paleness, F (L)

0 (0)

0.6 (4.3)

0 (0)

0 (0)

0.8 (5.0)(a)

Thymus: reddish foci, F (L)

0 (0)

0 (0)

0 (0)

0.8 (6.3)

0 (0)(a)

F      : fetal incidence.

L      : litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

(a)     : maximum incidence.

(b)     : mean incidence.

 

Fetal soft tissue malformations

Dose-level (mg/kg/day)

       0

    100

    400

  1000

HCD

Dams with live fetuses

     24

      23

      24

      16

       387

Number of live fetuses

   171

    165

    175

    118

     2404

Litters affected, n (%)

0 (0)

0 (0)

0 (0)

1 (6.3)

7 (1.8)(b)

Fetuses affected, n (%)

0 (0)

0 (0)

0 (0)

1 (0.8)

13 (0.5)(b)

Marked dilated ureter, F (L)

0 (0)

0 (0)

0 (0)

0.8 (6.3)

4.8 (4.8)(a)

F      : fetal incidence.

L      : litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

(a)     : maximum incidence.

(b)     : mean incidence.

 

Fetal incidences of cartilage

Dose-level (mg/kg/day)

        0

     100

   400

    1000

HCD

Dams with live fetuses

       24

       23

     24

        16

        388

Number of live fetuses

     185

     175

   182

      126

      2596

Cartilage of metatarsal
bone present, F(L)

7.6 (37.5)

6.3 (34.8)

10.4 (41.7)

25.4#(50.0)

36.8 (72.7)(a)

Cartilage of caudal
vertebra(e) present, F(L)

3.2 (16.7)

2.9 (21.7)

1.1 (8.3)

18.3#(31.3)

2.2 (15.0)(a)

Cartilage of sternebra(e) present, F(L)

1.6 (12.5)

2.3 (8.7)

0.5 (4.2)

11.9#(25.0)

3.8 (21.7)(a)

F      : fetal incidence.

L      : litter incidence.

Statistically significant;#: p < 0.001 for the number of fetuses affected.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

(a)     : maximum incidence.

(b)     : mean incidence.

 

Fetal incidences of skeletal

Dose-level (mg/kg/day)

        0

     100

   400

   1000

HCD

Dams with live fetuses

       24

       23

     24

       16

        388

Number of live fetuses

     185

     175

   182

     126

      2596

Hyoïd: unossified, F (L)

0.5 (4.2)

0.6 (4.3)

1.6 (8.3)

4.0* (12.5)

1.3 (5.0)(a)

Caudal vertebra(e): incomplete ossification of arch, F(L)

1.1 (8.3)

1.1 (8.7)

0 (0)

7.9**(25.0)

2.3 (15.0)(a)

Caudal vertebra(e): incomplete ossification of centrum, F(L)

1.1 (8.3)

2.3 (17.4)

0.5 (4.2)

8.7** (18.8)

1.4 (10.0)(a)

Caudal vertebra(e): unossified centrum, F(L)

0 (0)

0 (0)

0 (0)

7.1#(18.8)

1.4 (5.6)(a)

6thsternebra: unossified, F (L)

0 (0)

0 (0)

0 (0)

4.0* (12.5)

0.7 (5.0)(a)

6thsternebra: incomplete ossification, F (L)

0 (0)

1.7 (4.3)

0 (0)

7.1#(12.5)

2.9 (16.7)(a)

Metatarsal bone: unossified

first metatarsal, F(L)

7.6 (37.5)

6.3 (34.8)

10.4(41.7)

23.0#(43.8)

36.8 (72.7)(a)

F      : fetal incidence.

L      : litter incidence.

Statistically significant; *: p < 0.05, **: p < 0.01 and #: p < 0.001 for the number of fetuses affected.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

(a)     : maximum incidence.

(b)     : mean incidence.

 

Fetal skeletal malformations

Dose-level (mg/kg/day)

       0

    100

   400

   1000

HCD

Dams with live fetuses

     24

      23

     24

       16

        388

Number of live fetuses

   185

    175

   182

     126

      2596

Litters affected, n (%)

1 (4.2)

0 (0)

0 (0)

2 (12.5)

15 (3.9)(b)

Fetuses affected, n (%)

1 (0.5)

0 (0)

0 (0)

4 (3.2)

18 (0.7)(b)

Interparietal: split, F(L)

0.5 (4.2)

0 (0)

0 (0)

0 (0)

/

Lumbar vertebra(e): absent, F(L)

0 (0)

0 (0)

0 (0)

2.4 (6.3)

1.3 (8.7)(a)

Fused sternebra(e), F(L)

0 (0)

0 (0)

0 (0)

0.8 (6.3)

1.3 (8.7)(a)

F      : fetal incidence.

L      : litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/       : not reported in Historical Control Data.

(a)     : maximum incidence.

(b)     : mean incidence.

 

Distribution of fetal malformations

Dose-level (mg/kg/day)

0

100

400

1000

HCD

External

Litters affected, n (%)

0 (0)

0 (0)

0 (0)

0 (0)

11 (2.8)(a)

Fetuses affected, n (%)

0 (0)

0 (0)

0 (0)

0 (0)

13 (0.3)(a)

Soft tissue

Litters affected, n (%)

0 (0)

0 (0)

0 (0)

1 (6.3)

7 (1.8)(a)

Fetuses affected, n (%)

0 (0)

0 (0)

0 (0)

1 (0.8)

13 (0.5)(a)

Skeletal

Litters affected, n (%)

1 (4.2)

0 (0)

0 (0)

2 (12.5)

15 (3.9)(a)

Fetuses affected, n (%)

1 (0.5)

0 (0)

0 (0)

4 (3.2)

18 (0.7)(a)

Total

Litters affected / evaluated (%)

1/24 (4.2)

0/23 (0)

0/24 (0)

2/16 (12.5)

/

Fetuses affected / evaluated (%)

1/356 (0.3)

 0/340 (0)

0/357 (0)

5/244 (2.0)

/

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

(a)     : mean incidence.

/       : not reported in HCD.

 

 

 

Conclusions:
The test item, Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (batch No. 1629110) was administered by gavage, once daily, from Days 6 to 20 p.c., inclusive, to pregnant Sprague-Dawley rats at the dose-levels of 100, 400 and 1000 mg/kg/day. The control group received the vehicle, Polyethylene Glycol 400 (PEG 400), under the same experimental conditions.

On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 400 mg/kg/day (based on clinical signs, food consumption, body weight gain and mortality at 1000 mg/kg/day),
- the NOAEL for embryo-fetal development was considered to be 400 mg/kg/day (based on fetal dysmorphogenesis at 1000 mg/kg/day) in a context of severe maternal toxicity.
Executive summary:

SUMMARY

The objective of this study was to evaluate the potential toxic effects of the test item, Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female ratsfrom implantation until the day before scheduled hysterectomy [Day 6 to Day 20 post-coitum (p.c.)inclusive].

This GLP study was carried out according to OECD test guideline No. 414 (22 January 2001).

 

Methods

 

Three groups of 24 time-mated female Sprague-Dawley rats received the test item,Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate(batch No. 1629110), by the oral route (gavage), at a dose-level of 100, 400 or 1000 mg/kg/day, once daily from Day 6 to Day 20 p.c.inclusive.Another group of 24 time-mated rats received the vehicle only,Polyethylene Glycol 400 (PEG 400),under the same experimental conditions, and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. The test item concentrations were determined in the dose formulations. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recordedat designated intervals. On Day 21p.c.,the females were sacrificed and a macroscopicpost-mortemexamination was performed. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities (including cartilage).

Samples of the macroscopic lesions were collected and preserved in 10% buffered formalin.

 

Results

Chemical analyses

The concentrations of the test item in the dose formulations (-9.1% to -2.8%) remained within an acceptable range of variations (± 15%) when compared to the nominal concentrations.

No test item was observed in the control dose formulation.

 

Pregnancy status

At study termination on Day 21p.c.,there were 24, 23, 24 and 16 females with live fetuses in the groups given 0, 100, 400 and 1000 mg/kg/day, respectively.

 

Mortality

At 1000 mg/kg/day, one pregnant female was found dead and seven pregnant females were prematurely sacrificed.These unscheduled deaths were considered to be test item treatment-related.

 

Clinical signs

The following test item treatment-related findings were observed:

.          from 100 mg/kg/day: ptyalism, round back and/or piloerection,

.          from 400 mg/kg/day: emaciated appearance in isolated animals,

.          at 1000 mg/kg/day: loud and/or abdominal breathing defined as endpoints, pallor of extremities and/or soft feces.

 


Body weight

At 1000 mg/kg/day and when compared with controls, lower mean body weight gain was observed on Days 9-12 p.c. (+18 g vs. +24 g in controls) and Days 15-18 p.c. (+20 g vs. + 42 g in controls, p < 0.001). These findings were considered to be test item treatment-related and adverse.

The terminal mean body weight was not affected on Day 21 p.c.

 

Food consumption

At 1000 mg/kg/day and when compared with controls, there was lower mean food consumption (-10%, p < 0.05) that correlated with lower mean body weight gain on Days 15-18 p.c. This finding was considered to be test-item treatment-related and adverse.

 

Necropsy and macroscopicpost-mortemexamination

Whitish coloration of stomach mucosa, dilatation of intestines, small thymus and/or enlargement of adrenals were observed in 1/24 females given 400 mg/kg/day and 2/16 females given 1000 mg/kg/day; these were considered to be test item treatment-related.

 

Net body weight change

At 1000 mg/kg/day and when compared with controls, a lower mean net body weight change (+35 g vs. +45 g in controls, not statistically significant) was noted.

 

Hysterectomy data

There were no test item treatment-related effects.

Fetal body weight and percentage of male fetuses

There was no toxicologically important effect on mean fetal body weight.

The sex ratio was not affected by the test item treatment at any dose-level.

 

Fetal examinations

External examination

There were no test item-related variations or malformations at external examination.

 

Soft tissue examination

At all dose-levels and when compared with controls, there were higher litter and fetal incidences of dilated ureter (considered to be a non-adverse finding).

At 1000 mg/kg/day, one malformed fetus had marked dilatation of the ureter, for which a relationship to the test item treatment cannot be ruled out.

 

Cartilage and skeletal examinations

At 1000 mg/kg/day and when compared with controls, there were higher litter and fetal incidences of fetuses with delayed ossification: hyoid (unossified), caudal vertebra(e)(unossified centrum and/or incomplete ossification of arch and/or centrum), 6th sternebra (unossified or incomplete ossification) and 1st metatarsal bone (unossified).

One litter contained one malformed fetus (fused sternebrae) and another one contained three malformed fetuses[absent lumbar vertebra(e)],for which a test item treatment-related effect cannot be excluded.


Conclusion 

The test item, Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (batch No. 1629110) was administered by gavage, once daily, from Days 6 to 20 p.c.,inclusive, to pregnant Sprague-Dawley rats at the dose-levels of 100, 400 and 1000 mg/kg/day.The control group received the vehicle, Polyethylene Glycol 400 (PEG 400), under the same experimental conditions. 

On the basis of the results obtained in this study:

.          the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 400 mg/kg/day (based on clinical signs, food consumption, body weight gain and mortality at 1000 mg/kg/day),

.          the NOAEL for embryo-fetal development was considered to be 400 mg/kg/day (based on fetal dysmorphogenesis at 1000 mg/kg/day)in a context of severe maternal toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Species:
rat
Quality of whole database:
OECD guideline 414 reprotoxicity screening study (Kr. 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

- Effects on fertility:

Based on the available data, Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate is not classified for fertility according to the Regulation (EC) No 1272/2008 and the Directive 67/548/EEC criteria.

- Effects on developmental toxicity:

Considering the absence of developmental toxicity observed in the OECD 422 study and the fact that the developmental effects observed in the OECD 414 study were observed only in a context of severe maternal toxicity and restricted to one litter instead of being spread between litters. It is considered that the malformations observed are not linked to specific reprotoxic effect of the Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate. Therefore, no classification for developmental toxicity is warranted.

Based on the available data, Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate is not classified for developmental toxicity according to the Regulation (EC) No 1272/2008 and the Directive 67/548/EEC criteria.