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Toxicological information

Toxicity to reproduction

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Administrative data

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.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2014

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: brownish liquid stored at room temperature
Details on test material:
- Name of test material (as cited in study report): Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate. ISOBUTYL ACID PHOSPHATE, IBAP
- Physical state: brownish liquid.
- Stability under test conditions: the test item is assumed to be stable by the sponsor.
- Storage condition of test material: room temperature.

Test animals

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.

Administration / exposure

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.
Doses / concentrations
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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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)

Effect levels (P0)

open allclose all
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)

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 examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

No remarks.

Applicant's summary and conclusion

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.