Phosphoric acid, butyl ester

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A read across to Reaction mass of methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate was performed (for read across justification please refer to attached document).

The NOAEL for maternal toxicity was considered to be 120 mg/kg bw/day. In combination with severe maternal toxicity observed at 450 mg/kg bw/d an increase in pup loss at birth and a decreased litter weight was noted in the high dose group. This developmental effects are considered to be caused by the maternal toxicity at 450 mg/kg bw/d. The developmental NOAEL is set at 120 mg/kg bw/d.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

13 September 2013 to 11 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 90 Wistar Hannover (Crl:WI(Glx/BRL/Han)IGSBR) rats (45 males and 45 virgin females), 9 to 10 weeks old and weighing 225 to 250 g for males and 176 to 200 g for females, were ordered from Charles River Italia S.p.A., Calco (Lecco), Italy.
After arrival, on 13 September 2012, the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. Body weight ranges were 238 to 253 g for males and 175 to 194 g for females. A health check was then performed by a veterinarian.
An acclimatisation period of approximately 2 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

Animal room controls were set to maintain temperature and relative humidity at 22°C  2°C and 55%  15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.

In-life data from. 13 September 2013 to 10 November 2013

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified water

Details on exposure:

The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume.

The required amount of the test item was dissolved in the vehicle, purified water. The formulations were prepared daily (concentrations of 10, 24 and 90 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.

Details on mating procedure:

Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The proposed formulation procedure for the test item was checked during the pre-treatment period in the range of 10 to 90 mg/mL by chemical analysis (concentration) to confirm that the method was acceptable. Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (95-105%).
Samples of the formulations, prepared on Weeks 1 and 6, were analysed to check the concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for concentration of solutions (95-105%)

Duration of treatment / exposure:

Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.

Frequency of treatment:

once a day

Remarks:

Doses / Concentrations:
50 mg/kg bw/d
Basis:
actual ingested

Remarks:

Doses / Concentrations:
120 mg/kg bw/d
Basis:
actual ingested

Remarks:

Doses / Concentrations:
450 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

4 group comprised 10 male and 10 female rats receive the test item at the dose levels of 0, 50, 120, 450 mg/kg/day

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected in consultation with the Sponsor based on information from preliminary studies.

Parental animals: Observations and examinations:

Mortality

Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Clinical signs

Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

Clinical observations (Functional Observation Battery Tests)

Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observation were performed for individuals animals.

Grip strength and sensory reactivity to stimuli

Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order.

Motor activity assessment (MA)

Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order

Body weight

Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum

Food consumption

The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

Clinical pathology investigations

As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters, if possible) randomly selected from each group, under condition of food deprivation.
The blood samples collected were divided into tubes as follows:

EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests

The measurements performed on blood samples are listed below:

Haematology

Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count
- Neutrophils
- Lymphocytes
- Eosinophils
- Basophils
- Monocytes
- Large unstained cells
Platelets

Coagulation tests

Prothrombin time

Clinical chemistry

Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

Urinalysis (only males)

At the same time interval of the clinical pathology investigations, individual overnight urine samples were also collected from the same animals under the same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.

Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:

Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors

Oestrous cyclicity (parental animals):

Vaginal smears

Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the following:

a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Sperm parameters (parental animals):

Parameters examined in male parental generations:
[testis weight, epididymis weight, morphologycal evaluation of the seminiferous epithelium (staging of spermatogenic cycle).

Litter observations:

Parturition and gestation length

A parturition check was performed from Day 20 to Day 25 post coitum.
Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring are first detected in the cage was considered Day 0 post partum.

Pups identification, weight and observation

As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1 and 4 post partum.
Pups killed or dying during the lactation period were weighed before the despatch to necropsy.
Observation was performed once daily for all litters.

Postmortem examinations (parental animals):

Parental animals sacrificed for humane reasons and those that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia.

Parental males:
The males were killed after the mating of all females.

Parental females:
The females with live pups were killed on Day 4 post partum.
One high dose female with all pus stillborn (animal no. 93560069) was killed on Day 0 post partum and another high dose female (animal no. 93560073) was killed for humane reasons on Day 0 post partum.
The females which did not give birth 25 days after positive identification of mating (animal nos. 93560019, 93560023, 93560079) were sacrificed on Days 26, 27 or 27 post coitum.

Necropsy

The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.

Females:
All females were examined also for the following:

a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea (pregnant animals).

Organ weights

From all animals completing the scheduled test period, the organs were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved

Samples of all the tissues were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).

Postmortem examinations (offspring):

All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities.
All live pups sacrificed at termination were examined for external abnormalities and sex confirmation by gonadal inspection

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups was assessed by the non-parametric version of the Williams test

Reproductive indices:

The following reproductive indices were calculated:

Males
Copulatory Index (%) = no. of animals mated / no. of animals paired x 100
Fertility Index (%) = no. of males which induced pregnancy / no. of males paired x 100


Females
Copulatory Index (%) = no. of animals mated / no. of animals paired x 100
Fertility Index (%) = no. of pregnant females /no. of females paired x 100

Males and females
Pre-coital Interval = Mean number of days between pairing and mating

Offspring viability indices:

Females

Pre-birth loss was calculated as a percentage from the formula:

(No. of visible implantations - total litter size at birth ) / No. of visible implantations x 100


Pup loss at birth was calculated as a percentage from the formula:

(Total litter size - live litter size) / Total litter size x 100


Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:

(Total litter size at birth - live litter size at Day 4) / Total litter size at birth x 100

Pre-implantation loss was calculated as a percentage from the formula:

(no. corpora lutea- no. implantations) / no. corpora lutea x 100

Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

three high dose females were found dead and 2 high dose females were sacrificed for humane reasons

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):

trachal changes suggetsed to be related to treatment

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Mortality and fate of females:
Three high dose females (nos. 93560063, 93560067 and 93560071) were found dead during the study, on Days 21 and 15 post coitum and Day 12 of treatment, respectively.
The cause of deaths is suggested to be potentially related to the irritant properties of the test compound.
In addition, 2 high dose females (nos. 93560073, 93560069) were sacrificed for humane reason on Day 0 post partum.

Clinical observations (Functional Observation Battery Tests), Neurotoxicity assessment (removal of animals from the home cage and open arena):
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

Clinical signs:
No significant clinical signs were noted in the males and in the surviving females, with the exception of one low dose not pregnant female, which showed piloerection, pallor and red staining in cage tray on Days 24/25 post coitum and one high dose pregnant female which showed hunched posture, respiratory distress and red staining on mounth and urogenital region (Day 0 post partum)

Body weights:
Body weights of males were unaffected by treatment.
No changes were observed in the body weight of females during pre-mating and post coitum periods. A very slight, not significant at statistical analysis, reduction in body weight was observed in the high dose females on Day 4 post partum when compared to controls (-10%).

Food consumption:
Food consumed was comparable between the control and treated groups.

Motor activity and sensory reactivity to stimuli:
No relevant differences in motor activity, grip strength and sensory reactivity to stimuli were observed.

Haematology:
No changes of toxicological significance were observed.
The statistically significant differences between males dosed with 50 mg/kg/day and controls (haematocrit and leucocytes) were considered to be incidental.
A slight increase of erythrocytes, haemoglobin and haematocrit and slight leucopenia were recorded in female no. 93560079. These findings were considered of no toxicological relevance.

Coagulation:
No changes were recorded.

Clinical chemistry:
Changes of a number of parameters, mainly metabolic markers, were observed in animals from all treated groups, with no dose-relation.
Males dosed with 450 mg/kg/day showed a slight increase of triglycerides (43%), phosphorus (19%) and bile acids (283%). Bile acids were also increased in some animals dosed with 50 and 120 mg/kg/day (165% and 65%, respectively), with no dose-relation.
In addition, animal no. 93560022 (50 mg/kg/day) showed moderate increase of transaminase enzymes. This finding was considered unrelated to treatment.
Treated females showed decrease of triglycerides (44% to 58%), cholesterol (18% to 28%), urea (20% to 22%), phosphorus (11% to 18%) and potassium (17% to 24%) and increase of glucose (10 to 27%).

Urinalysis only males:
No changes were recorded.

Terminal body weight and organ weights:
No treatment-related changes were observed in the weight of the organs in either sexes.
Terminal body weight was unaffected by treatment

Macroscopic observations:
No treatment-related changes were noted.

Microscopic observations:
In a single female rat treated with the high dose, tracheal minimal subchronic inflammation in the submucosa associated with minimal mucosal hyperplasia, was noted. The tracheal changes are suggested to be related to the irritant properties of the test compound.

Spermatogenic cycle:
Evaluation of the spermatogenic cycle did not show differences between the groups. Regular layering in the germinal epithelium was noted.

Key result

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: pregnant females

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

trachea

other: mortality

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

At birth % pup loss increased in the high dose group with a consequent reduction in live litter size

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight decrease in litter weight in the high dose group

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups :
At birth, % pup loss was considerably increased in the high dose group with respect to the controls, although without a statistical significance. Consequently, a reduction of approximately 30% in live litter size was detected in the same group.
Slight decrease in litter weight was also detected in the high dose group on Days 1 and 4 post partum of approximately 14 and 17 %, respectively.
No significant differences in sex ratio were detected.

Clinical signs of pups :
The signs noted in treated pups were considered incidental since similar to those detected in control pups.

Necropsy findings in decedent or humane killed pups and in pups sacrificed on Day 4 post partum:
No milk in stomach was observed at necropsy in the decedent and humane killed pups of control and treated groups.
No abnormalities were found in pups of control and treated groups sacrificed on Day 4.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mortality at highest dose observed

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

450 mg/kg bw/day (actual dose received)

System:

other: decreased mean litterweight and pup viability caused by maternal toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

450 mg/kg bw/day (actual dose received)

Treatment related:

yes

Oestrus cycle – Before pairing - Group summary data

 

 

 -----------------------------------------------------------------------------------------------------------------------------------

           Group 1              Group 2             Group 3             Group 4

 Animal   Oestrus    Animal   Oestrus   Animal   Oestrus   Animal   Oestrus

 Number   Cycles     Number   Cycles    Number   Cycles    Number   Cycles

 -----------------------------------------------------------------------------------------------------------------------------------

 93560001    1      93560021    3      93560041    2      93560061    3

 93560003    4      93560023    3      93560043    2      93560063    2

 93560005    4      93560025    3      93560045    2      93560065    2

 93560007    3      93560027    3      93560047    2      93560067    3

 93560009    4      93560029    3      93560049    3      93560069    2

 93560011    3      93560031    3      93560051    3      93560071    2

 93560013    3      93560033    4      93560053    3      93560073    4

 93560015    3      93560035    2      93560055    2      93560075    4

 93560017    5      93560037    2      93560057    3      93560077    2

 93560019    1      93560039    2      93560059    3      93560079    4

 

     Means  3.1                 2.8                 2.5                 2.8

 -----------------------------------------------------------------------------------------------------------------------------------

 Note: The number of oestrus cycles is based on the number of non sequential days the dams were in oestrus.

Reproductive parameters of males - Summary data

 -----------------------------------------------------------------------------------------------------------------------------------

                              Group      1          2         3          4

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Copulatory Index%                    100.0      100.0     100.0      100.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Fertility Index%                      90.0       90.0     100.0       88.9

 

 -----------------------------------------------------------------------------------------------------------------------------------

Reproductive parameters of females - Summary data

 -----------------------------------------------------------------------------------------------------------------------------------

                              Group      1          2         3          4

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Copulatory Index%                    100.0      100.0     100.0      100.0

 

 -----------------------------------------------------------------------------------------------------------------------------------

 

   Fertility Index%                      90.0       90.0     100.0       88.9

 

 -----------------------------------------------------------------------------------------------------------------------------------

Conclusions:

A read across to Reaction mass of methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate was performed (for read across justification please refer to attached document).
The NOAEL for maternal toxicity was considered to be 120 mg/kg bw/day. In combination with severe maternal toxicity observed at 450 mg/kg bw/d an increase in pup loss at birth and a decreased litter weight was noted in the high dose group. This developmental effects are considered to be caused by the maternal toxicity at 450 mg/kg bw/d. The developmental NOAEL is set at 120 mg/kg bw/d.

Executive summary:

A read across to Reaction mass of methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate was performed (for read across justification please refer to attached document).

Study design 

The toxic effects on rats of both sexes after repeated dosing with Reaction mass of methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring were investigated.

The vehicle waspurified water. All doses were administered at a constant volume of 5 mL/kg body weight.

 

Group Number	Treatment (mg/kg/day)	Number of animals
1 2 3 4	0 50 120 450	10M+10F 10M+10F 10M+10F 10M+10F

 

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32/33 days.

Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3post partumor the day before necropsy.

The following investigations were performed in all groups: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology, clinical chemistry and males urinalysis), litter weight, pups observations, macroscopic observations and organ weights.

External examination for pups at Day 4 of lactation and external and internal examination in pups found dead were also performed. 

The histopathological examination was performed on control and high dose groups (five males and five females randomly selected). The examination included also the identification of the stages of the spermatogenic cycle.

 

Mortality and fate of females 

A total of 3 high dose females were found dead during the study and the cause of death is suggested to be potentially related to the irritant properties of the test compound.In addition, 2 high dose females were sacrificed for humane reasons on Day 0post partum.

A total of 3 females were found not pregnant at necropsy.

The number of females with live pups on Day 4post partumwas: 9 in each of the control and low dose groups, 10 in the mid-dose group and 4 in the high dose group.

 

Daily clinical signsand weekly clinical observations (Functional ObservationBatteryTests)

No treatment-related clinical signs were noted in the males and in the surviving females.

Weekly functional observation battery tests were unaffected by treatment.

 

Body weight and body weight gain 

Body weights of males were unaffected by treatment.

No changes of toxicological relevance were observed in the body weight of females.

 

Food consumption 

No effects on food consumption were observed.

 

Motor activity and sensory reactivity to stimuli 

No differences of toxicological significance were seen.

 

Haematology 

No changes of toxicological significance were seen.

 

Coagulation

No changes were recorded.

 

Clinical chemistry 

Changes of a number of parameters, mainly metabolic markers, were observed in animals from all treated groups, generally with no dose-relation, and/or with opposite trend in the two sexes.

 

Urinalysis – males only 

No changes were observed.

 

Oestrous cycle, mating performance and reproductive parameters 

All surviving females mated. No treatment-related anomalies were noted in the oestrous cycle of the treated females when compared to controls.

The copulatory and fertility indices were similar among groups.

Pre-coital interval and the number of copulation plugs were unaffected by treatment.

 

Implantation, pre-birth loss data and gestation length of females 

No significant differences were found in the number of corpora lutea, implantations, gestation length and total litter size between control and treated groups.

An increase in pre-birth loss % was noted in mid- and high dose groups with respect to the controls.

 

Litter data and sex ratio of pups 

At birth, % pup loss was considerably increased in the high dose group with respect to the controls with a consequent reduction in live litter size. In addition, a slight decrease in litter weight was also detected in the high dose group on Days 1 and 4post partum.

No significant differences in sex ratio were detected.

 

Clinical signs of pups 

Clinical signs of pups were comparable between treated and control groups. 

Necropsy findings in decedent or humane killed pups and in pups sacrificed on Day 4post partum 

No milk in stomach was observed at necropsy in the decedent and humane killed pups of control and treated groups.

No abnormalities were found in pups of control and treated groups sacrificed on Day 4.

 

Terminal body weight and organ weights 

No treatment-related changes were observed in the weight of the organs in either sexes.

Terminal body weight was unaffected by treatment.

 

Macroscopic observations 

No treatment-related changes were noted.

 

Microscopic observations 

In a single female rat treated with the high dose, tracheal minimal subchronic inflammation in the submucosa associated with minimal mucosal hyperplasia, was noted. The tracheal changes are suggested to be related to the irritant properties of the test compound.

 

Spermatogenic cycle

Evaluation of the spermatogenic cycle did not show differences between the groups. Regular layering in the germinal epithelium was noted.

 

Conclusions

 The NOAEL for maternal toxicity was considered to be 120 mg/kg bw/day. In combination with severe maternal toxicity observed at 450 mg/kg bw/d an increase in pup loss at birth and a decreased litter weight was noted in the high dose group. This developmental effects are considered to be caused by the maternal toxicity at 450 mg/kg bw/d. The developmental NOAEL is set at 120 mg/kg bw/d

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

All studies used for read across are reliable without restriction.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

1. A read across to Reaction mass of methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate was performed (for read across justification please refer to IUCLID Chapter 13).
Study design
The toxic effects on rats of both sexes after repeated dosing with Reaction mass of methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring were investigated.The vehicle waspurified water. All doses were administered at a constant volume of 5 mL/kg body weight. 

Group Number	Treatment (mg/kg/day)	Number of animals
1 2 3 4	0 50 120 450	10M+10F 10M+10F 10M+10F 10M+10F

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32/33 days.Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3post partumor the day before necropsy.The following investigations were performed in all groups: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology, clinical chemistry and males urinalysis), litter weight, pups observations, macroscopic observations and organ weights. External examination for pups at Day 4 of lactation and external and internal examination in pups found dead were also performed. The histopathological examination was performed on control and high dose groups (five males and five females randomly selected). The examination included also the identification of the stages of the spermatogenic cycle.

Mortality and fate of females 

A total of 3 high dose females were found dead during the study and the cause of death is suggested to be potentially related to the irritant properties of the test compound.In addition, 2 high dose females were sacrificed for humane reasons on Day 0post partum.A total of 3 females were found not pregnant at necropsy.The number of females with live pups on Day 4post partumwas: 9 in each of the control and low dose groups, 10 in the mid-dose group and 4 in the high dose group. 

Daily clinical signsand weekly clinical observations (Functional Observation Battery Tests)

No treatment-related clinical signs were noted in the males and in the surviving females.

Weekly functional observation battery tests were unaffected by treatment.

 Body weight and body weight gain 

Body weights of males were unaffected by treatment.No changes of toxicological relevance were observed in the body weight of females.

Food consumption 

No effects on food consumption were observed.

Motor activity and sensory reactivity to stimuli 

No differences of toxicological significance were seen.

Haematology 

No changes of toxicological significance were seen.

Coagulation

No changes were recorded.

Clinical chemistry 

Changes of a number of parameters, mainly metabolic markers, were observed in animals from all treated groups, generally with no dose-relation, and/or with opposite trend in the two sexes.

Urinalysis – males only 

No changes were observed.

Oestrous cycle, mating performance and reproductive parameters 

All surviving females mated. No treatment-related anomalies were noted in the oestrous cycle of the treated females when compared to controls.

The copulatory and fertility indices were similar among groups.

Pre-coital interval and the number of copulation plugs were unaffected by treatment.

Implantation, pre-birth loss data and gestation length of females 

No significant differences were found in the number of corpora lutea, implantations, gestation length and total litter size between control and treated groups.

An increase in pre-birth loss % was noted in mid- and high dose groups with respect to the controls.

Litter data and sex ratio of pups 

At birth, % pup loss was considerably increased in the high dose group with respect to the controls with a consequent reduction in live litter size. In addition, a slight decrease in litter weight was also detected in the high dose group on Days 1 and 4post partum.

No significant differences in sex ratio were detected.

Clinical signs of pups 

Clinical signs of pups were comparable between treated and control groups. 

Necropsy findings in decedent or humane killed pups and in pups sacrificed on Day 4post partum 

No milk in stomach was observed at necropsy in the decedent and humane killed pups of control and treated groups.

No abnormalities were found in pups of control and treated groups sacrificed on Day 4.

Terminal body weight and organ weights 

No treatment-related changes were observed in the weight of the organs in either sexes.

Terminal body weight was unaffected by treatment.

Macroscopic observations 

No treatment-related changes were noted.

Microscopic observations 

In a single female rat treated with the high dose, tracheal minimal subchronic inflammation in the submucosa associated with minimal mucosal hyperplasia, was noted. The tracheal changes are suggested to be related to the irritant properties of the test compound.

Spermatogenic cycle

Evaluation of the spermatogenic cycle did not show differences between the groups. Regular layering in the germinal epithelium was noted.

Conclusions

The NOAEL for maternal toxicity was considered to be 120 mg/kg bw/day. In combination with severe maternal toxicity observed at 450 mg/kg bw/d an increase in pup loss at birth and a decreased litter weight was noted in the high dose group. These developmental effects are considered to be caused by the maternal toxicity at 450 mg/kg bw/d. The developmental NOAEL is set at 120 mg/kg bw/d

 

2. A second read across to Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol is performed (for read across justification please refer to IUCLID Chapter 13). The aim of this study was to assess the possible effects of Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol on male and female fertility and embryofetal development after repeated dose administration inWistar rats. The test item was administered daily in graduated doses to 3 groups of test animals. Animals of an additional control group were handled identically as the dose groups but received aqua ad injectionem (sterile water), the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistar rats. During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals. Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males and females from each group. Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment and at the end of the study. Epididymal sperm motility and testicular sperm head count was evaluated in all male animals. After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. A full histopathological evaluation of the tissues was performed on 5 randomly selected male and female animals of the control and high dose groups. Organs showing gross alterations were also examined histopathologically. The examinations of these organs were extended to animals of the medium and low dose groups if treatment-related changes were observed in high dose groups. The following doses were evaluated:
Control:                       0        mg/kg body weight
Low Dose:                   50       mg/kg body weight
Medium Dose:             200     mg/kg body weight
High Dose:                  500     mg/kg body weight
Summary Results
2 male animals of the HD group as well as 4 female animals of the HD group died/were euthanized due to morbidity during the treatment period.
For male HD animals, an attenuated body weight gain was observed during the pre-mating period. In female animals body weight was significantly lower in HD group when compared to C group (p<0.01). Furthermore, body weight gain was significantly attenuated in HD group between GD0 and GD7 (p<0.05). In addition, body weight gain was attenuated during the whole pre-mating and gestation period in HD animals. In males statistical significant decreased food consumption was found during pre-mating days 7-14 which was also found as a tendency during pre-mating days 1-7. In females statistical significant decreased food consumption was evaluated in HD group during GD 7-14.
No treatment-related effect on litter data was observed such as the total number of pups born, number of male and females, sex ratio, live pups on PND 0 and PND 4. No treatment-related effect on litter data was found in any of the dosing groups when compared to the C group. No treatment-related effect was observed during the pre-coital interval or during the duration of gestation when compared with the control group. All pregnancies resulted in normal births. The group mean numbers of corpora lutea, number of implantation sites, number of live pups born on PND 0, percentage of pre-implantation loss and post-implantation loss remained unaffected due to the treatment with test item when compared with the control group. No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls. No treatment-related gross external findings were observed in any of the treated groups. At the end of the treatment period, no influence of the test item could be found on sperm motility or sperm head counts. In female animals, weights of uteri (with cervix) showed a slight tendency to a dose dependent decrease. This could not be confirmed by relative weights (to body weight). Since no statistical significance could be calculated, since the relative uterus weights did not confirm the absolute weights and since no clear histopathological findings could be detected, a test item relation could not be clearly mentioned. Hence, a toxicological relevance within this study cannot be assumed. There was no indication of test item-related histopathological findings in reproductive organs of the surviving male or female rats of this study. The reproductive organs of the females found non-gravid at terminal sacrifice showed normal reproductive sexual cycle. Minimal tracheal changes in two surviving animals treated at 500 mg/kg/day were considered to be most probably related to a local irritant effect of the test item formulation.
Thus, the NOAEL for male and female animals in this study is considered to be 200 mg/kg body weight.

3. A third read across to Phosphoric acid, hexadecyl ester is performed (for read across justification please refer to IUCLID Chapter 13).
Four groups of 11 males and 11 females were treated by gavage with the test item once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum.
The following dose levels were used:
Group 1:      0 mg/kg body weight/day (control group)
Group 2: 100 mg/kg body weight/day
Group 3: 300 mg/kg body weight/day
Group 4: 1000 mg/kg body weight/day
A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water (ELGA)). The following results were obtained:
MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS 
All animals survived the scheduled study period. No test item-related clinical signs were noted at any dose level.
FOOD CONSUMPTION OF PARENTAL ANIMALS 
Food consumption was not affected by the treatment with the test item at any dose level.
BODY WEIGHTS OF PARENTAL ANIMALS 
Body weights and body weight gains of males and females were not affected by the treatment up to and including the dose level of 1000 mg/kg body weight/day.
REPRODUCTION AND BREEDING DATA 
No effects on mating performance, fertility, corpora lutea count, duration of gestation, post-implantation loss, litter size or breeding loss were observed at any dose level.
CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS 
No test item-related effects were found on hemathology or biochemistry parameters in males or females.
ORGAN WEIGHTS OF PARENTAL ANIMALS 
At 1000 mg/kg body weight/day, there was a statistically significantly reduced thymus weight in males measured which was considered to be incidental since it was within the historical background range. SPERM ANALYSES IN PARENTAL MALES 
Sperm analysis did not reveal any test item-related effects.
MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATION OF PARENTAL ANIMALS 
Macroscopical and microscopical examinations did not reveal any test item-related effects.
FINDINGS IN PUPS AT FIRST LITTER CHECK 
No test item-related findings were noted in pups at any dose level.  Pups sex ratio was not affected by the exposure to the test item at any dose level.
PUP WEIGHTS TO DAY 4 POST PARTUM 
No effects on pup body weights or body weight gain were noted at any dose level.
MACROSCOPICAL FINDINGS IN PUPS 
No test item-related findings were noted in pups at any dose level.
CONCLUSION 
Based on these results, NOEL (No Observed Effect Level) for general toxicity in males and females was considered to be 1000 mg/kg body weight/day. The NOEL for reproduction/developmental toxicity was considered to be 1000 mg/kg body weight/day.

4. 20 rats (10 males and 10 females) per dose were treated with Dibutyl phosphate (containing 19.1% triester) at dose levels of 0, 30, 100, 300 or 1000 mg/kg bw/d for a period of a minimum of 44 days (males). This study was performed according to OECD 422. The females were mated and allowed to breed and pups were sacrificed and macrospically examined on day 4 post partum. Hematology and clinical biochemistry were performed in males. Both males and females were examined macroscopically. Histology was performed in selected organs in the control and high dose group.
With regard to the reproduction of the parental males and females, no significant difference to the control group was observed for any of the indicators examined, even in the 1000 mg/kg bw/d group. One animal in the high dose group had a difficult delivery where all pups died, anf therefore the gestation index tended to be sightly low, but there were no significant changes. In the 1000 mg7kg bw/d group the incidence of females whose pups all died was increased (3 animals), and so the number of live pups on day 4 and the vialbility tended to be low. In the parental females whose pups all died, erosion of the stomach were observed and severe pathological changes confirmed, but no abnormalities of the pituitary or reproductive organs were reported. The changes in these developmental toxcity indicators in the parental females are therefore deemed to have been secondary general toxicological effects.
In all groups unrelated to dose, occasionally a female did not become pregnant depsite copulation having been confirmed. These animals exhibited no pathological changes indicative of abnormal reproductive capapcity, so this was thought to have been accidental.
Since the tendency to a low pup viability is considered to be caused by the maternal toxicity at 1000 mg/kg bw/d and with reference to the different identity of the substance used in this described study (Dibutyl phosphate, containing 19.1% triester, labelled as carcinogenic) and Phosphoric acid, butyl ester (CAS No. 12788-93-1; not containing Dibutyl phophate triester) no reliable NOAEL (developmental/reproductive) could be derived from this OECD 422 study.

Short description of key information:
A read across to an OECD 422 study performed with Reaction mass of methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate was performed (for read across justification please refer to IUCLID Chapter 13).
A total of 3 high dose females were found dead during the study and the cause of death is suggested to be potentially related to the irritant properties of the test compound .In addition, 2 high dose females were sacrificed for humane reasons on Day 0 post partum. At birth, % pup loss was considerably increased in the high dose group with respect to the controls with a consequent reduction in live litter size. In addition, a slight decrease in litter weight was also detected in the high dose group on Days 1 and 4 post partum.
The NOAEL for maternal toxicity was considered to be 120 mg/kg bw/day. In combination with severe maternal toxicity observed at 450 mg/kg bw/d an increase in pup loss at birth and a decreased litter weight was noted in the high dose group. These developmental effects are considered to be caused by the maternal toxicity at 450 mg/kg bw/d. The developmental NOAEL is set at 120 mg/kg bw/d.

Justification for selection of Effect on fertility via oral route:
The OECD 422 study performed with Reaction mass of methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate was selected as relevant reproductive toxicity screening study due to its reliability and since it providesthe most sensitive NOEL.

Justification for selection of Effect on fertility via inhalation route:
In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the inhalation route because exposure of human via inhalation, especially in a higher extent than via oral application as performed in the animal studies, is considered unlikely taking into account the vapour pressure of the substance and the physical form (viscous liquid). Furthermore, an acute toxicity inhalation study did not reveal any systemic effects in rats.

Justification for selection of Effect on fertility via dermal route:
In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the dermal route because
- no systemic effects or other evidence of absorption were observed in skin and eye irritation, as well as in acute dermal toxicity studies in rabbits
- only local effects in gastrointestinal tract were reported after repeated oral uptake of Phosphoric acid, butyl ester and its structural analogues in the course of OECD 422 studies in rats.
Therefore, due its corrosive properties only local skin effects, no systemic toxic changes are expected to occur.

Effects on developmental toxicity

Description of key information

The test item, Hordaphos MDB dissolved in refined groundnut oil (Arachis oil) was administered once daily by oral gavage to three treatment groups of twenty four pregnant female Wistar rats each from day 5 to day 19 of gestation at dose levels of 50, 200 and 400 mg/kg body weight/day.A control group of twenty four females was administered with vehicle (refinedgroundnut oil) alone.

The treatment with test item resulted in no mortalities. All the dams survived till the scheduled sacrifice.

No test item related clinical signs were observed in any of the pregnant female animals of low, intermediate and high dose groups as well as in control group. The body weight and feed consumption during gestation period in all treatment groups were comparable with thre control group. The mean body weight gain (%) was slightly reduced in the high dose animals on gestation days 14 and 17.

No treatment-related effects were observed in reproduction parameters such as litter weight, corpora lutea count, early and late resorption and number of live and dead foetuses, pre-implantation loss, post-implantation loss as well as sex ratio at any dose level in the female animals treated with 50, 200 and 400 mg/kg body weight/day. The mean fetal weight of high dose pups was slightly decreased.The external and visceral variations observed in foetuses were randomly distributed across the groups. Therefore, these findings were considered as incidental findings and not test item-related.

Type and distribution of variations noted during skeletal examination at the dose levels of 50, 200 and 400 mg/kg body weight/day did not indicate any test item-related effects.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept 2017 - May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Jan 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding, RCC Laboratories India Private Limited, Genome Valley, Turkapally, Shameerpet (Mandal), Ranga Reddy District, Hyderabad - 500 078 India
- Age at study initiation: 12 -14 weeks
- Weight at study initiation: 242.1 - 282.6 g
- Housing: During acclimatization and randomization period all animals were housed in groups of two in polycarbonate cages (approximate internal dimensions of 365 mm x 202 mm x 180 mm height) with corn cob bedding. After randomization, males and females were housed individually. During the mating phase, animals were housed on one male: one female basis within each dose group.
- Diet (e.g. ad libitum): Teklad Certified Global 14% Protein Rodent Maintenance Diet (Lot Number: 2014C-040117MA) from ENVIGO was provided ad libitum.
- Water (e.g. ad libitum): Aquaguard filtered tap water was provided ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1°C to 22.3°C
- Humidity (%): 56 to 66%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 7. Nov. To: 19. Dec. 2017

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on preliminary solubility testing
- Concentration in vehicle: 5, 20 and 40 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas chromatography
Samples of the dose formulations from one set per dose group was taken immediately after preparation once before commencement of dosing (i.e. on day 5 of gestation and on day 19 of gestation (days were considered from first dosing of the group) for homogeneity and on day 11 of gestation for dose concentration (mean of homogeneity given as dose concentration). Analyses were performed.
The test item in corn oil was assayed on three occasions
01 Stability of test item at 0th, 2nd and 4th hour in corn oil matrix for the fortified concentrations of 5 mg/mL and 40 mg/mL.
02 Initial homogeneity test on Top, mid and bottom layers of corn oil fortified with of test item Viz., 5 mg/mL (Low dose), 20 mg/mL (Intermediate dose), and 40 mg/mL (High dose)
03 Dose concentration of test item in corn oil fortified with 5 mg/mL (Low dose), 20 mg/mL (Intermediate dose), and 40 mg/mL (High dose).
04 Final homogeneity test on Top, mid and bottom layers of corn oil fortified with of test item Viz., 5 mg/mL (Low dose), 20 mg/mL (Intermediate dose), and 40 mg/mL (High dose). Calibration solution, CS5 was used as bracketing standard.

Details on mating procedure:

Animals were paired on a one male: one female basis for a maximum period of fourteen days. In case pairing is unsuccessful, re-mating of female with proven male was considered. Each female was examined for vaginal smear or the presence of a copulation plug in the vagina. The presence of sperm in the vaginal smear and vaginal plug was taken as positive evidence of mating (Day 0 of gestation).

Duration of treatment / exposure:

day 5 of gestation and continued until day 19 of gestation

Frequency of treatment:

daily

Duration of test:

mating period + gestation period until gestation day 20

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
based on results of preliminary performed studies

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
twice daily for mortality
once daily for clinical signs

BODY WEIGHT: Yes
acclimatisation period: weekly
premating period: on first day of mating and weekly thereafter
gestation: on gestation days 0, 3, 5, 8, 11, 14, 17 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
On gestation day 3, 5, 8, 11, 14, 17 and 20


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
All female animals were weighed
- Organs examined:
The uteri of females were examined for the presence and number of implantation sites and the number of corpora lutea in the ovaries were determined

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
Gravid uteri including the cervix were weighed

- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes
The foetuses were removed, identified, weighed, sexed and evaluated for external malformation/variation. Each fetus was subject to external examination, which included all visible structures, surfaces and orifices (including the oral cavity)

- Soft tissue examinations: Yes
One-half of the fetuses (alternating foetuses within the litter) independent of sex were processed for visceral (soft tissue) alterations. All the foetuses selected for visceral examination were observed by Staple’s dissection technique for visceral malformations/alterations and discarded. The organs and structures of the head, neck, thorax and abdomen were observed. Organs and structures of live foetuses like skin, palate, nasal cavity, brain, eyeballs, olfactory bulb, lateral ventricle, internal ear, spinal cord, oesophagus, trachea, thoracic cavity, diaphragm, thyroid gland, thymus, lung, heart (blood vessels, atrium, ventricles, etc), liver, spleen, pancreas, stomach, intestinal tract, kidney, adrenal, ureters, urinary bladder and reproductive organs were observed under stereomicroscope for any malformations/variations.

- Skeletal examinations: Yes
One-half of the fetuses (alternating foetuses within the litter) independent of sex were processed for skeletal alterations. All the foetuses selected for skeletal examination were eviscerated, processed, stained with Alizarin red S (single staining) and examined for skeletal malformations/alterations by using stereomicroscope. All the bone structures of the head, spine, rib cage, pelvis and limbs were observed.

Statistics:

The following statistical methods were used to analyze the body weight, body weight change, feed consumption, reproduction and external, visceral and skeletal alterations/variations:
Data was summarized in tabular form. Statistical analysis was performed using Statplus program. All the data was checked for Normality with Shapiro-Wilk W test and for Homogeneity with Bartlett Chi-Square test. Each group of animals was subjected to Analysis of Variance (ANOVA) among the groups, and Bonferroni Test for unequal replications. For discontinuous data, nonparametric test (Mann-Whitney U-Test) was used. Values are given as mean ± standard deviation (SD).

Indices:

Pre–implantation loss (%)
(Number of Corpora Lutea - number of implantation sites)/ Number of Corpora Lutea x100
Post–implantation loss (%)
(Number of implantation sites - Total number of live foetuses)/ Number of implantation sites x 100
Sex Ratio (% males)
Number of male foetuses (Day 0) / Total number of foetuses (Day 0) x 100
Variation Incidence (%)
Number of foetuses with variation/ Total Number of foetuses examined x100

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No significant difference in the body weight were observed in any of the low, intermediate and high dose group animals when compared with control group, whereas the body weight gain (%) was significantly decreased in the intermediate and high dose group when compared to control group on day 14 and 17 of gestation. These changes could not be attributed to toxicity due to test item exposure since, there were no change in absolute body weight of dam even though increase in body weight gain in percentage on day 20.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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:

no effects observed

Description (incidence and severity):

Weight of cervix and gravid uterus did not show significant difference between the control and treated groups.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

please refer to description of findings

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:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Details on maternal toxic effects:

no changes reported

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no adverse changes up to highest dose tested observed

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No significant difference observed in litter weight in all treatment groups as compared to control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

External examination of foetuses revealed small cranium head in one foetus of control group. Small size and pale body was observed in one pub of group 1 (dam no. 27) and in two pubs each in group 2 (dam no. 34 and 36), group 3 (dam no. 55 and 62) and group 4 (dam no. 95 and 96). Additionally, in one pub of dam no. 96 (group 4) head abnormalities (absence of eye bulge, Naris atresia Nose, absence of tongue, absence of mandible, absence of oral opening and smaller upper jaw in mouth) were observed. The findings of small size and pale bodies are not dose related and in line with historical control data. Likewise, the head abnormalities in one single pub out of a total of 208 pubs are within historical control ranges and hence considered a spontaneous finding without toxicological relevance.
No significant difference was observed in external findings in all treatment groups when compared with control group.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal examination of foetuses revealed non ossified sternal center twenty seven in control, twelve in low and intermediate, twenty six in high dose group. While non ossified Xyphoid six in control, five in low and nine in high dose group were observed.
Type and distribution of variations noted during skeletal examination at the dose levels of 50, 200 and 400 mg/kg body weight did not indicate any test item-related effects.

Visceral malformations:

no effects observed

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

no changes reported

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed up to highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

MORTALITY AND CLINICAL SIGNS

	Group
	1	2	3	4
Dose (mg/kg bw)	0	50	200	400
Mortality	0/24	0/24	0/24	0/24
Clinical signs	0/24	0/24	0/24	0/24

Expressed as number of animals affected/Total number of animals

  SUMMARY OF BODY WEIGHT

BODY WEIGHTS (G) – SUMMARY - FEMALE

Group	Body weights (in gram)
	Gestation Day
		0	3	5	8	11	14	17	20
Group 1	Mean	248.0	253.0	257.7	266.4	276.8	287.7	299.3	319.2
	SD	8.9	9.8	10.6	11.4	11.5	11.7	13.0	17.2
	Min	232.8	237.4	242.1	251.4	258.6	265.1	269.1	272.6
	Max	266.1	270.4	276.4	286.5	299.4	312.2	328.9	354.5
	N	24	24	24	24	24	24	24	24
Group 2	Mean	250.8	255.6	260.7	268.5	277.7	287.0	298.9	319.7
	SD	7.8	7.9	8.4	8.9	11.1	12.4	14.0	18.7
	Min	236.4	240.1	246.3	254.8	263.7	268.4	271.6	275.8
	Max	268.4	274.2	282.6	290.5	302.3	315.6	325.9	348.6
	N	24	24	24	24	24	24	24	24
Group 3	Mean	250.4	255.5	260.2	267.0	275.8	285.3	296.9	318.5
	SD	8.9	9.0	9.4	10.4	12.2	12.1	12.8	16.4
	Min	239.2	243.1	246.4	251.8	259.5	270.0	273.9	276.5
	Max	268.5	272.1	275.8	284.9	297.3	308.9	322.8	345.2
	N	24	24	24	24	24	24	24	24
Group 4	Mean	250.5	255.4	260.7	267.6	275.5	283.9	294.5	315.1
	SD	7.6	7.8	8.3	9.5	10.0	10.4	11.5	17.4
	Min	236.8	242.1	248.8	256.5	263.8	268.9	272.3	275.8
	Max	265.9	271.2	278.9	290.6	298.4	306.5	315.3	338.2
	N	24	24	24	24	24	24	24	24

BODY WEIGHT GAIN (%) – SUMMARY

FEMALE

Group	Body weight Gain %
	Gestation Day
		0	3	5	8	11	14	20
Group 1	Mean	0.0	2.0	3.9	7.4	11.6	16.0	28.7
	SD	0.0	0.7	0.9	1.3	1.8	2.3	5.8
	N	24	24	24	24	24	24	24
Group 2	Mean	0.0	1.9	4.0	7.1	10.7	14.4	27.5
	SD	0.0	0.5	1.0	1.6	2.6	3.1	6.3
	N	24	24	24	24	24	24	24
Group 3	Mean	0.0	2.1	3.9	6.7	10.2*	13.9*	27.2
	SD	0.0	0.4	0.7	1.2	2.2	2.6	5.4
	N	24	24	24	24	24	24	24
Group 4	Mean	0.0	2.0	4.1	6.8	10.0*	13.4*	15.8
	SD	0.0	0.4	0.8	1.3	1.7	2.4	6.8
	N	24	24	24	24	24	24	24

* Significant at p ≤ 0.05 level with g

PREGNANCY STATUS

GROUP	G1	G2	G3	G4
DOSE (mg/kg bw)	0	50	200	400
FEMALE ANIMALS MATED	24	24	24	24
PREGNANT FEMALE ANIMALS	21	21	22	20

MACROSCOPIC FINDINGS

GROUP	G1	G2	G3	G4
DOSE (mg/kg bw)	0	50	200	400
ANIMALS EXAMINED	24	24	24	24
ANIMALS WITHOUT ABNORMALITY	24	24	24	24
ANIMALS AFFECTED	0	0	0	0

SUMMARY OFGRAVID UTERINE WEIGHT

Group	G1	G2	G3	G4
Dose (mg/kg bw)	0	50	200	400
No. of Dams	21	21	22	20
Mean	54.99	53.18	56.63	52.38
SD	19.64	19.13	20.27	14.55

  SUMMARY OF MATERNAL DATA

Group		No. of CL	Live Implants	Dead Implants	Early Resorption[w1]	Late Resorption	Combined Implants	Pre Implantation Loss (%)	Post Implantation Loss (%)
G1	Total	275	212	1	12	4	229	364	209
	Mean	13.10	10.10	0.05	0.57	0.19	10.90	17.34	9.94
	SD	2.32	3.58	0.22	0.81	0.87	3.39	22.73	14.86
G2	Total	276	214	0	4	1	219	429	147
	Mean	13.14	10.19	0.00	0.19	0.05	10.43	20.43	7.02
	SD	2.46	3.82	0.00	0.40	0.22	3.63	24.97	21.94
G3	Total	322	235	1	12	1	249	511	156
	Mean	14.00	10.22	0.04	0.52	0.04	10.83	22.21	7.11
	SD	2.04	4.40	0.21	0.95	0.21	4.42	30.31	16.69
G4	Total	278	208	0	9	1	218	403	70
	Mean	13.90	10.40	0.00	0.45	0.05	10.90	20.16	3.50
	SD	2.17	3.03	0.00	1.15	0.22	3.43	24.21	7.77

 

 SUMMARY OF LITTER DATA

Group		Mean Foetuses Weight (g)	Mean Total Litter Weight (g)	Total Number Foetuses	Male Foetuses (%)	Female Foetuses (%)
Control	Mean	3.59	36.49	212	51.7	48.3
	SD	0.77	16.48
	N	21	21
Low Dose	Mean	3.48	36.72	214	43.9	56.1
	SD	0.58	11.22
	N	20	20
Medium Dose	Mean	3.50	36.09	235	50.6	49.4
	SD	0.77	14.63
	N	22	22
High Dose	Mean	3.37	33.75	208	55.8	44.2
	SD	0.84	10.11
	N	20	20

Expressed values are statistically not significant at p ≤ 0.05. All values within historical control range.

 SUMMARY OF EXTERNAL FINDINGS

Group		G1	G2	G3	G4
Dose (mg/kg bw)		0	50	200	400
No. of Dam Examined		24 (21)	24 (21)	24 (22)	24 (20)
No. of Foetuses Examined		210	213	235	208
Variation Incidence – Number (%)
No. of Foetus with Variations	Small in size	0(0.00)	3(1.41)	1(0.43)	2(0.96)
	Head small cranium	1(0.48)	0(0.00)	0(0.0)	0(0.0)
	Absent eye bulge	0(0.0)	1(0.53)	0(0.0)	1(0.48)
	Nose: Naris atresia	0(0.0)	0(0.0)	0(0.00)	1(0.48)
	Body pale	0(0.0)	3(1.41)	1(0.43)	1(0.48)
	Mouth: Absence of tongue	0(0.0)	0(0.0)	0(0.00)	1(0.48)
	Mouth: Absence of oral opening	0(0.0)	0(0.0)	0(0.00)	1(0.48)
	Mouth: Smaller upper jaw	0(0.0)	0(0.0)	0(0.00)	1(0.48)

All observed head abnormalities are related to one single pub (foetus no. 2 of dam no. 96) of group 4.

  SUMMARY OF VISCERAL FINDINGS

Group		G1	G2	G3	G4
Dose (mg/kg bw)		0	50	200	400
No. of Litter Examined		21	21	22	20
No. of Foetuses Examined		100	103	112	101
Variation Incidence – Number (%)
No. of Foetus with Variations	Ureters: Convoluted	1(1.0)	1(0.97)	0(0.0)	0(0.0)

SUMMARY OF SKELETAL FINDINGS

Group	G1	G2	G3	G4
Dose (mg/kg bw)	0	50	200	400
No. of Dam Examined	21	21	22	20
No. of Foetuses Examined	110	110	123	107
Variation Incidence – Number (%)
Sternal Centers (5)
Not Ossified	27(24.55)	12(10.91)	12(9.76)	26(24.3)
Xyphiod
Not Ossified	6(5.45)	5(4.55)	0(0.00)	9(8.41)

HISTORICAL CONTROL DATA

Type of study – Prenatal and Developmental Toxicity Studies
Species – Wistar Rat

No of studies – 7
Total no of pups - 1231

Serial No.	Mean Foetus Weight (g)	No. of Litters (Dam)	Number of Foetus
1	37.1831 ± 9.735	10	99
2	32.6552 ± 13.545	20	193
3	25.9775 ± 10.873	18	142
4	27.6178 ± 14.556	21	163
5	36.7318 ± 16.766	21	212
6	40.0196 ± 16.206	22	244
7	28.0719 ± 10.401	20	178

Type of study – Prenatal and Developmental Toxicity Studies
Species – Wistar Rat

No of studies – 7                    
Total no of pups - 1171

Foetal abnormalities	Incidence
Small in size	18
Domed head	1
Head – small cranium	2
Spleen – small in size	8
Pale spleen	5
Uterus - convoluted	6
Pale body	3
Nose - misshapen	1
Absence of tongue and mandible	1

Conclusions:

Based on the findings from this developmental toxicity study, it is considered that the test item is non teratogenic in rats and the NOAEL for teratogenicity of the test item can be fixed as 400 mg/kg bw/day, under the present experimental conditions. The NOAEL for maternal and foetal toxicity was considered as 400 mg/kg bw/day.

Executive summary:

The test item, Hordaphos MDB dissolved in refined groundnut oil (Arachis oil) was administered once daily by oral gavage to three treatment groups of twenty four pregnant female Wistar rats each from day 5 to day 19 of gestation at dose levels of 50, 200 and 400 mg/kg body weight/day. A control group of twenty four females was administered with vehicle (refined groundnut oil) alone.

The treatment with test item resulted in no mortalities. All the dams survived till the scheduled sacrifice.

No test item related clinical signs were observed in any of the pregnant female animals of low, intermediate and high dose groups as well as in control group. The body weight and feed consumption during gestation period in all treatment groups were comparable with thre control group. The mean body weight gain (%) was slightly reduced in the high dose animals on gestation days 14 and 17.

No treatment-related effects were observed in reproduction parameters such as litter weight, corpora lutea count, early and late resorption and number of live and dead foetuses, pre-implantation loss, post-implantation loss as well as sex ratio at any dose level in the female animals treated with 50, 200 and 400 mg/kg body weight/day. The mean fetal weight of high dose pups was slightly decreased. The external and visceral variations observed in foetuses were randomly distributed across the groups. Therefore, these findings were considered as incidental findings and not test item-related.

Type and distribution of variations noted during skeletal examination at the dose levels of 50, 200 and 400 mg/kg body weight/day did not indicate any test item-related effects.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restriction

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

no further data mandatory

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action the effects observed in animals are regarded as relevant for humans.

Justification for classification or non-classification

There is no evidence to suggest that a classification for reproductive toxicity is appropriate.

With reference to the OECD 422 studies performed with Phosphoric acid, butyl ester and its structural surrogates, Phosphoric acid mixed esters with butyl alcohol and ethylene glycol, reaction mass of methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate and reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphate and

and the lack of teratogenic effects in an OECD 414 study performed with phosphoric acid, butyl ester, it is concluded that the registration substance is not subject to classification and labelling according Regulation 1272/2008/EC regarding reproductive toxicity.