Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
The effects on rats of both sexes after repeated dosing with the test substance, as well as any effects of the test item on male and female reproductive preformance, such as gonadal function, conception, parturition and early lactation of the offspring were investigated. Due to mortality in the high dose group the NOAEL for maternal toxicity was considered to be 120 mg/kg bw/d. 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. Thus, 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:
experimental study
Adequacy of study:
key study
Study period:
13 September 2013 to 11 November 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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:
other: 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
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:
50 mg/kg bw/d
Basis:
actual ingested
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:
120 mg/kg bw/d
Basis:
actual ingested
Dose / conc.:
450 mg/kg bw/day (actual dose received)
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:
histopathology: non-neoplastic
other: pregnant females in pregnant females
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (actual dose received)
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:
respiratory system: upper respiratory tract
Organ:
trachea
Treatment related:
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: effects observed are considered to be secondary caused by significant maternal toxicity at 450 mg/kg bw/d
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

-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:
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.
Executive summary:

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 partum or 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 0 post partum.

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

The number of females with live pups on Day 4 post partum was: 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 4 post 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

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:
Reliable without restrictions.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

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. Thess 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:
This OECD 422 study was selected as relevant reproductive toxicity screening study due to its reliability and since it provides a 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).

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 were observed in skin and eye irritation studies in rabbits
- only local effects in trachea and gastrointestinal tract were reported after repeated oral uptake in the course of an OECD 422 study 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

An OECD 414 study was performd with the test substance. The main findings of the study are:

- There were no mortalities, clinical signs or gross necropsy findings at any of the dose tested.

- Mean body weights and gais, food consumption were comparable across the tested doses.

- Mean values of maternal paramaters comprising uterine weight and number of corpora lutea, implantation, early deaths, late deaths, pre-implantation loss and post implantation loss were statistically comparable to the vehicle control group up to the highest dose of 300 mg/kg bw/d.

- The litter parameters comprising total number of fetuses, number of live fetuses, male and female fetal weights, anogenital distance in male and female fetuses were statistically comparable to vehicle control group up to the highest tested dose of 300 mg/kg bw/d.

- Fetal external, visceral and skeletal examination revealed no signs of teratogenicity up to the highest dose of 300 mg/kg bw/d.

- Thyroid hormaone profile (T3, T4 and TSH), thyroid gland weights and histopathology of thyroid gland were unaffected by treatment with the test item up to the highest dose of 300 mg/kg bw/d.

In conclusion, based on the above findings, the NOAEL for maternal toxicity and fetal developmental toxicity is 300 mg/kg bw/d.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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)
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 - Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana, India
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation analysis was carried out from samples prior to the initiation of treatment and before the termination.

The prepared formulations was sampled in duplicate sets wherein one set was used for analysis and another was kept as back up set which was stored at room temperature in the experimental room. For each set, composite samples were drawn in three triplicates for each dose formulation. In case of control, duplicate composite samples were drawn. Dose formulations were sent for formulation analysis to determine the concentration of the test item using a validated analytical method (Study No.: G16490).
Formulations were considered acceptable if the overall mean result (calculated using all the 3 replicate values) is within ± 15.0 % of the claimed concentration and the relative standard deviation (% RSD, calculated using all the 3 replicate values) is equal to or less than 10.0 %.

The back up samples were either used for reanalysis when the results of first set of analysis were outside the acceptable limits or discarded if the results of first set of analysis were within the acceptable limits
Details on mating procedure:
Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 12 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy
Duration of treatment / exposure:
15 days
Frequency of treatment:
Gestation day 5 to Gestation day 19
Dose / conc.:
50 mg/kg bw/day
Remarks:
Group 2 (Low dose)
Dose / conc.:
120 mg/kg bw/day
Remarks:
Group 3 (Mid dose)
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 3 (High dose)
No. of animals per sex per dose:
Day '0" Pregnant rats : 24
Control animals:
yes, concurrent vehicle
Details on study design:
Dose Selection rationale
Based on the dose range finding study carried out by the sponsor at the doses of 100, 300 and 1000 mg/kg/day, the dose levels of 50 (G2), 120 (G3) and 300 (G4) mg/kg/day have been selected for this study in consultation with the Sponsor.

Vehicle control - 0 mg/kg/day
Low dose - 50 mg/kg/day
Mid dose - 120 mg/kg/day
High dose - 300 mg/kg/day
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day (pre dose and post dose)
- Cage side observations checked in table [No.2] were included
- At necropsy Thyroid gland was collected and weighed and subjected to histopathology
- Thyroid harmone analysis [T3, T4, TSH]
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Gross evaluation of placenta: Yes
Fetal examinations:
External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
- Ano-genital distance: Yes: [all per litter]
- Fetus sex (external determination based on anogenital distance and internal sex based on gonadal examination)
Statistics:
The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances are heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences are found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female were analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external, visceral and skeletal observations for variations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon (Mann-Whitney) pairwise comparison of the treated groups with the control group was performed, when the group differences were significant.

The incidence of dams with and without resorptions was tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.

Statistically significant differences (p<0.05) were designated as * throughout the report
Historical control data:
Refer: Annexure 7 iin the Report
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs findings at any of the doses tested.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities at any of the doses tested
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and gains were comparable across the tested doses.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption were comparable across the tested doses.
Clinical biochemistry findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid gland weights were unaffected by treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross necropsy findings at any of the doses tested
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathology of thyroid gland were unaffected by treatment.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone profile (T3, T4 and TSH) were unaffected by treatment.
Number of abortions:
no effects observed
Description (incidence and severity):
Mean values of maternal parameters comprising uterine weight and number of corpora lutea, implantations, early deaths, late deaths,
pre-implantation loss and post implantation loss were statistically comparable to the vehicle control group up to the highest dose of
300 mg/kg/day
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre-implantation loss and post implantation loss were statistically comparable to the vehicle control group up to the highest dose of
300 mg/kg/day
Early or late resorptions:
no effects observed
Description (incidence and severity):
Early deaths and late deaths were statistically comparable to the vehicle control group up to the highest dose of
300 mg/kg/day
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
maternal abnormalities
other: Teratogeneicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Male and female fetal weights were statistically comparable to vehicle control group up to the highest tested dose of 300 mg/kg/day
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
Fetal external examination revealed no signs of teratogenicity up to the highest tested dose of 300 mg/kg/day
Skeletal malformations:
no effects observed
Description (incidence and severity):
Fetal skeletal examination revealed no signs of teratogenicity up to the highest tested dose of 300 mg/kg/day
Visceral malformations:
no effects observed
Description (incidence and severity):
Fetall visceral examination revealed no signs of teratogenicity up to the highest tested dose of 300 mg/kg/day
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: ne effects observed up to the highest dose tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on the findings of the OECD 414 study, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and fetal developmental toxicity is 300 mg/kg/day (highest dose tested).
Executive summary:

An OECD 414 study was performed with the test substance. Ninety six presumed pregnant Wistar rats were assigned to four groups bybody weight stratification (Group 1 to Group 4) and each group (G1: control, G2: low dose, G3: mid dose and G4: high dose) consisted of 24 presumed pregnant rats (gestation day 0). Day `0' of gestation for each individual female rat in the study was considered as the day on which vaginal smear was found positive for sperm.

The test substance in vehicle (Milli-Q water) was administered at 0, 50, 120 and 300 mg/kg/day. The control group received the vehicle only. A constant dose volume of 10 mL/kg body weight was administered to all groups.

All rats were observed for clinical signs, morbidity and mortality, body weight changes and food consumption. Prior to caesarean section, blood was collected for Thyroid hormone analysis. At caesarean section on GD 20, dams were examined for gross pathological changes and thyroid gland was collected, weighed and subjected to histopathological examination. The uterus was removed by laparotomy, weighed and the contents were examined for number of implantation sites, early and late resorptions and number of fetuses. The number of corpora lutea in ovaries was counted. All the fetuses were sexed, weighed and examined for external malformations. Approximately half the number of fetuses from each dam was examined for visceral malformations and the remaining half was evaluated for skeletal malformations.

The main findings of the study are presented below:

- There were no mortalities, clinical signs or gross necropsy findings at any of the doses tested.

- Mean body weights and gains, food consumption were comparable across the tested doses.

- Mean values of maternal parameters comprising uterine weight and number of corpora lutea, implantations, early deaths, late deaths, pre-implantation loss and post implantation loss were statistically comparable to the vehicle control group up to the highest dose of 300 mg/kg/day.

- The litter parameters comprising total number of fetuses, number of live fetuses, male and female fetal weights, anogenital distance in male and female fetuses were statistically comparable to vehicle control group up to the highest tested dose of 300 mg/kg/day.

- Fetal external, visceral and skeletal examination revealed no signs of teratogenicity up to the highest tested dose of 300 mg/kg/day.

- Thyroid hormone profile (T3, T4 and TSH), thyroid gland weights and histopathology of thyroid gland were unaffected by treatment with the test item up to the highest dose of 300 mg/kg/day.

In conclusion, based on the above findings, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and fetal developmental toxicity is 300 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable without restrictions
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/developmental toxicity is appropriate.

With reference to the OECD 422 and OECD 414 studies performed with the test item, the substance not subject to classification and labelling according to 1272/2008/EC regarding reproductive/developmental toxicity.

Additional information