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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
other: Data sharing dispute
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Phosphoric acid, mixed esters with Bu alc. and ethylene glycol
EC Number:
284-716-0
EC Name:
Phosphoric acid, mixed esters with Bu alc. and ethylene glycol
Cas Number:
84962-20-9
IUPAC Name:
Reaction mass of Phosphoric acid mono-(2-hydroxy-ethyl) ester and Phosphoric acid monobutyl ester and Phosphoric acid bis-(2-hydroxy-ethyl) ester

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Sex: male and female; the female animals were non-pregnant and nulliparous.
- Age at the start of the treatment period: males: 9-10 weeks old, females: 9-10 weeks old.
- Body weight at the allocation of the animals to the experimental groups: males: 238 – 271 g (mean: 252.63 g, ± 20% = 202.10 – 303.15 g); females: 161 – 188 g (mean: 173.88 g, ± 20% = 139.10 – 208.65 g)
- The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfarethe animals were bred for experimental purposes.
- Housing and Feeding Conditions: Full barrier in an air-conditioned room
- Temperature: 22 +- 3°C
- Relative humidity: 55 +- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10/h
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0939)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 030512)
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimised. In case of unsuccessful mating, re-mating of females withproven males of the same group was considered.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each dosing concentration was analysed for nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations. Samples for the nominal concentration verification were taken from all groups in study week 1 (first week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) (16 samples in total). Samples for homogeneity were taken from the top, middle and bottom of the high dose, medium dose, and the low dose preparation in study week 1 and 5 (18 samples in total). Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the preparation (at room temperature), from high, medium and low dose preparations (6 samples in total). All formulation samples were analysed on the day of sample collection were stored at -20° C were analysed after completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 122543 according to a phase plan which was amended to the study plan.
Duration of treatment / exposure:
The test item is orally administered daily, i.e. 7 days per week in graduated doses to several groups of test animals (male and female), one dose level per group with a maximum exposure of 54 days in total (at least 14 days pre-mating, up to 14 days mating, 22 days of gestation and 4 days of post-partum). Duration of treatment in males was maximally 29 days.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:

- Dose selection rationale:
The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL. The doses were selected on the basis of data from a Dose Range Finding Study (BSL study no. 120216).
- Rationale for animal assignment (if not random): Random
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males from each group and between days 1-3 of the lactation period in 5 randomly selected females from each group outside the home cage using a functional observational battery of tests

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

OTHER:
- Parameters of heamatology, clinical biochemistry, and urine were investigated from 5 randomly selected male and female animals at terminal sacrifice.
Oestrous cyclicity (parental animals):
No examination
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations: testis weight, epididymis weight, sperm count in testes, sperm motility, histopathology of testes and epididymides
Litter observations:
PARAMETERS EXAMINED
- The following parameters were examined in [F1] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross external anomalies, weight gain.

GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [At application day 28 or 29]
- Maternal animals: All surviving animals [at Lactation Day 4.]

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ Weights
The wet weight of the below described organs from 5 randomly selected animals / sex / group was recorded as soon as possible. Paired organs were weighed separately. In addition reproductive organs of all animals were weighed.
Organs Weighed at Necropsy for the randomly selected animals:
liver; uterus with cervix; kidneys; thymus; adrenals; thyroid/parathyroid glands; testes; spleen; epididymides; brain; prostate, seminal vesicles and coagulating glands; pituitary glands; ovaries; heart

Histopathology
All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the above mentioned organs (See below) preserved and evaluated histologically. A full histopathology was carried out on the preserved organs and tissues of 5 randomly selected male and female animals of the control and high dose groups which were sacrificed at the end of the treatment period. These examinations were extended to animals of all other dosage groups for treatment-related changes that were observed in the high dose group. Hence, trachea, lung, and stomach were examined in LD and MD groups. Testes (unilateral), epididymides (unilateral), ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) and all organs showing gross lesions were examined in all animals of all groups. For the testes (unilateral), a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
brain (cerebrum, cerebellum and pons); heart; spinal cord; ovaries (females); liver; uterus with cervix (females); kidneys ; vagina (females); adrenal glands; testes (unilateral; males); stomach; epididymides (unilateral; males); small and large intestines (including Peyer´s patches); prostate and seminal vesicles with coagulating glands as a whole (males); thymus; urinary bladder; thyroid lymphnodes (mesentric and axillary); spleen peripheral nerve (e.g. sciatic nerve) with skeletal muscle; lung and trachea; bone with bone marrow (sternum); mammary glands; pituitary glands; skin; oesophagus; gross lesions
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at Lactation Day 4.

GROSS NECROPSY
- Gross necropsy consisted of external examinations.
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant)
Reproductive indices:
Copulation index; fertility index, delivery index were calculated.
Offspring viability indices:
Viability index was calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Several clinical symptoms were found in male and female animls which could be clearly attributed to the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male HD animals as well as female treatment groups effects were found on body weight decelopment. In male HD as well as female MD and HD groups, an affected food consumption was evaluated.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Several changes in organ weights were observed in male and female animals.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Several findings were observed in organs of male and female animals but no test item-related histopathological findings in reproductive organs of the surviving male or female rats of this study were evaluated.
Other effects:
not examined

Reproductive function / performance (P0)

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

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: effects reported

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
act. ingr.
Remarks:
maternal dose
Sex:
male/female
Basis for effect level:
other: effects reported

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

P0 animals

Mortality
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. The male animals were found dead/euthanized on mating and post-mating days 8 and 15. The female animals were found dead/euthanized on Premating Days 13 (twice) as well as gestation days 17 and 19. No mortality occurred in the other treatment or control groups. 

Clinical Observations
Several clinical symptoms were found which could be clearly attributed to the test item. In male treatment animals e.g. vocalization (4/10 HD animals), moving the bedding (3/10 HD animals; 1/10 MD animal), abnormal breathing (9/10 HD animals, 1/10 MD animal), slight piloerection (10/10 HD animals, 7/10 MD animals, 1/10 LD animals), moderate piloerection (8/10 HD animals, 4/10 MD animals, 1/10 LD animals) were found increased when compared with C group. In female treatment animals e.g. vocalization (5/10 HD animals), half eyelid closure (2/10 HD animals), moving the bedding (8/10 HD animals), abnormal breathing (10/10 HD animals), moderate salivation (3/10 HD animals, 1/10 MD animals), slight salivation (4/10 HD animals, 1/10 MD animals, 1/10 LD animals), slight piloerection (10/10 HD animals, 9/10 MD animals, 2/10 LD animals), moderate piloerection (10/10 HD animals, 1/10 MD animals, 1/10 LD animals), severe piloerection (4/10 HD animals) were observed. One female animal of the LD group bite the cannula off and swallowed it. This animal was observed with a high frequency in order to detect abnormalities due to the swallowed cannula. 

Functional Observations
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. Body temperature of female HD animals was slightly increased at the end of the treatment. This could be treatment related and of toxicological relevance. 

Body Weight Development
In male animals body weight increase was comparable for C, LD, and MD animals during the treatment period. For male HD animals, an attenuated body weight gain was observed during the pre-mating period but not during the mating and post-mating period. This attenuated body weight gain can be attributed to the test item and is assumed to be of toxicological relevance. 
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). Furthermore, body weight gain was attenuated during the whole pre-mating and gestation period in HD animals. A tendency to attenuation could be also mentioned for LD and MD groups during the gestation period. However, for LD and MD groups a test item relation cannot be clearly considered. For HD group a test item relation can be mentioned which is assumed to be toxicological relevant. 

Food Consumption
In correlation to the body weight and body weight change, the food consumption in both males and females showed some differences in treatment groups when compared with C group. 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. Again, this was found as a tendency during the whole gestation period. For MD animals a tendency to decreased food consumption was also found during the gestation period. 
Since the decreased food consumption of the HD animals correlates with an attenuated body weight gain this is considered to be test item related and of toxicological relevance. A test item relation can be also mentioned for MD animals which cannot be clearly stated to be toxicological relevant. 

Precoital Interval and Duration of Gestation
No treatment-related effect was observed during the pre-coital interval or during the duration of gestation when compared with the control group. The values were comparable between the groups. All pregnancies resulted in normal births.
Successful mating resulted in 9, 8, 10 pregnancies in the control, low, and medium dose groups, respectively. From the 6 surviving animals of the HD group, 5 were found to be pregnant. 

Pre- and Post-Natal Data
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. Although there was an increase in the percentage of pre-implantation loss of the LD group as well as of post-implantation loss in the MD group compared to the controls, due to the lack of statistical significance and dose-dependency, this effect cannot be attributed to the treatment.

Reproductive Indices
A reduced copulation index (when compared to C animals) was detected for LD, MD, and HD animals. Respective 90 % could be found. This is not assumed to be test item related. Fertility index, delivery index, as well as viability index were not different when compared to C animals. 

Haematology and Coagulation
At the end of the treatment period a slight tendency towards an increased amount of white blood cells could be observed in male HD animals (6.04 x 103/µl) when compared to C animals (4.68 x 103/µl). No statistical significance as well as no dose dependency could be detected. Hence, this increase is not assumed to be test item related. 
In female animals, an increased percentage of monocytes could be observed. This increase was dose dependent but not statistical significant. 1.88 % (C ), 2.48 % (LD), 5.78 (MD), and 12.98 % (HD) were measured. The HD value is above to what could be expected when comparing with our historical C data. This increased amount of monocytes could be test item related as well as toxicological relevant in the HD group.
Percentage of eosinophils of female animals was significantly increased in MD animals (0.83 %) when compared to C animals (0.24 %). Furthermore, percentage of eosinophils in HD group was slightly but not significantly increased with 0.65 %). Since this data is still in the range of our historical C data and since not dose dependency could be found it is not assumed to be test item related. 
All other values of haematology and of coagulation are not changed when comparing treatment groups and C group. 

Clinical Biochemistry
At the end of the treatment period levels of Cholesterol were significantly increased in male LD group (1.17; p<0.05) and HD group (1.18; p<0.05) when compared with C group (0.86). Animals of MD group exhibited a Chol value of 0.98.Values of LD and HD group were above to what could be expected regarding our historical C data. Although no clear dose dependency was detected, these increased values could be test item related and may indicate liver or kidneys as target organ. However, due to the absence of clear other findings (e.g. histopathology) and due to the mild characteristics of the increase a toxicological relevance is not assumed.
Levels of TBA were significantly increased (p<0.05) in male LD group when compared with C group. 20.26 as well as 9.3 could be measured, respectively. Since no increase was found for MD and HD group, this is assumed to be not test item related. 
In female animals, values of SGOT (ASAT) were slightly (p>0.05) decreased in treatment groups. 47.0 (C ), 38.7 (LD), 39.1 (MD), as well as 35.2 (HD) were measured. These slight decreases were not statistically significant and not dose dependent. A test item relation cannot be excluded but seems unlikely. Changes in serum levels of ASAT may indicate a liver, heart, or muscle disturbance. However, this is related to increased but not decreased values. Furthermore, since no other parameter indicates a disturbance of these organs/tissues a toxicological relevance is not assumed. 
Values of Phosphate are slightly decreased in HD group (1.74) when compared to C group (3.21). This decrease was not statistical significant. Several reasons exist for slightly low phosphate levels and the irritant effects of the test item could be e.g. responsible for a decreased absorption of phosphate from the food. However, since no other clear indications for the reason of the decreased phosphate levels are detected and since the decrease is not statistical significant it is assumed that this decrease may be test item related but is without toxicological relevance within this study. 

Urinalysis
The urinalysis performed in male animals revealed no test- item related effect in any of the treatment groups compared to the control group.
The urine analysis in female animals revealed increased blood content in HD animals as well as an increased protein content in treatment groups. Taken both things together, this may indicate the kidneys as a target organ. However, since no systematic finding was detected during histopathology and clinical biochemistry and since no organ weight changes were measured the test item relation and toxicological relevance seems unlikely. Especially in case of the blood content, contaminations during the necropsy could be responsible for this. 

Evaluation of Sperm Count and Sperm Motility
At the end of the treatment period, no influence of the test item could be found on sperm motility or sperm head counts. The treatment groups were comparable to the C group and showed values which could be expected for healthy animals of this age. 

Pathology
Few specific gross pathological changes were recorded for the male and female animals of the HD group which were assumed to be test item related and of toxicological relevance.
E.g. a gased gastro-intestinal tract could be mentioned in 4/10 female HD animals as well as 2/10 male HD animals. A small sized spleen or thymus was found in 2/10 female HD animals. 

Organ Weight
For a detailed description see Table 33 to Table 36 and Table 69 to Table 72.
In male animals absolute weight of spleens was slightly increased in MD groups and was significantly increased (p<0.05) in HD group. The weights were 0.614 g (C), 0.722 g (MD) and 0.809 g (HD). This could be partly confirmed by weights in relation to body weight where 0.173 % (C ), 0.197 %(MD), and 0.228 % (HD) were mentioned. In female animals, absolute weights of spleens of HD group were slightly increased when compared to C group. In particular, 0.732 g as well as 0.647 g could be measured, respectively. This could be confirmed by relative spleen weights of female animals, Respective 0.307 % (HD) as well as 0.269 % (C ) were found. Increased spleen weights could be an indicator of an immune stimulation. However, no clear pattern could be found during the histopathological analysis. Since histopathology is kown to be a more sensitive parameter than simple weight measurements and since spleen weights are known for a relatively high interanimal variability a toxicological relevance seems unlikely but however cannot be excluded for the male HD group. 

Absolute prostate weights (plus seminal vesicles and coagulating glands) was decreased in male HD group (1.89 g) when compared to C group (2.2 g). This could be confirmed by relative weights (to body weight) and 0.63 g (C ) as well as 0.54 g (HD) could be measured. Due to the high variability as well as the absent histopathological findings a test item relation is not assumed. 
In male animals, absolute weights of thymus were slightly increased in HD group (0.43 g) when compared to C group (0.39 g). Relative organ weights of male animals showed no alteration. In female animals, absolute weights of thymus were slightly decreased. When compared with C group (0.272 g), a decrease towards 0.227 g(LD), 0.255 g (MD), and 0.203 g (HD) were evaluated. This could be confirmed by relative thymus weights (to body weight). 0.113 % (C ); 0.096 % (LD), 0.103 % (MD), 0.085 % (HD) were calculated. Due to the mile characteristics of the increase (female) and decease (male) a test item relation cannot be clearly stated. A toxicological relevance within this study cannot be considered. 
In female animals, weights of uteri (with cervix) showed a slight tendency to a dose dependent decrease. As absolute weights 0.686 g (C ), 0.654 g (LD), 0.604 (MD), and 0.598 (HD) were measured. 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 couldnot be clearly mentioned. Hence, a toxicological relevance within this study cannot be assumed. 

Histopathology
Two males and four females treated at 500 mg/kg/day died or were sacrificed moribund during the treatment period, all of which showed gaseous distension of the gastrointestinal tract. Among these, male No. 40, found dead, had prominent findings in the respiratory system, indicative of gavaging error or regurgitation/aspiration of test item formulation into the airways. The other five decedents showed combinations of histological lesions in the gastrointestinal and/or respiratory tract which were indicative of a local irritant effect of the test item formulation. Degenerative/atrophic changes of the lymphoid organs and hypocellularity of the bone marrow in all of these animals were considered to be secondary to bad general condition and/or agonal stress. There was no clear indication of a direct toxic effect of the test item on any of the organs evaluated in these animals, including the reproductive organs.
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. No directly test item-related histopathological findings were noted in the other organs evaluated in this study. 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.

F1 animals

Litter Data
No treatment-related effect of the 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.

Litter Weight Data
No treatment-related effect on litter data was found in any of the dosing groups when compared to the C group.

Pup Survival Data
No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls. 

Pup External Findings
No treatment-related gross external findings were observed in any of the treated groups. Few incidences of external findings were observed in all groups (e.g. dark snout) which were considered to be spontaneous and not related to the test item.

Applicant's summary and conclusion

Conclusions:
A read across to Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol was performed. At a dose level of 500 mg/kg body weight mortalities occurred in male and female animals. Thus, the NOAEL for male and female animals in this study is considered to be 200 mg/kg body weight. There were no treatment related changes observed for reproductive and developmental parameters. Hence, the dose 500 mg/ kg body weight / day (highest tested dose level) is assumed to be NOEL for reproductive/ developmental toxicity.
Executive summary:

A read across to Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol was performed.

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 in Wistar 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 female Wistar 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. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. 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.

The males were sacrificed after completion of the mating period on treatment days 29 and 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26.

Pups sacrificed on post natal day 4 and those found dead, were carefully examined for gross external abnormalities.

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

The test item formulation was prepared freshly on each day of administration. The test item was dissolved inaqua ad injectionemand administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28-30 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministration volume was 5 mL/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.

Several clinical symptoms were found which could be clearly attributed to the test item. In male treatment animals e.g. vocalization (4/10 HD animals), moving the bedding (3/10 HD animals; 1/10 MD animal), abnormal breathing (9/10 HD animals, 1/10 MD animal), slight piloerection (10/10 HD animals, 7/10 MD animals, 1/10 LD animals), moderate piloerection (8/10 HD animals, 4/10 MD animals, 1/10 LD animals) were found increased when compared with C group.

In female treatment animals e.g. vocalization (5/10 HD animals), half eyelid closure (2/10 HD animals), moving the bedding (8/10 HD animals), abnormal breathing (10/10 HD animals), moderate salivation (3/10 HD animals, 1/10 MD animals), slight salivation (4/10 HD animals, 1/10 MD animals, 1/10 LD animals), slight piloerection (10/10 HD animals, 9/10 MD animals, 2/10 LD animals), moderate piloerection (10/10 HD animals, 1/10 MD animals, 1/10 LD animals), severe piloerection (4/10 HD animals) were observed. For both, male and female animals, these clinical symptoms are assumed to be toxicological relevant in HD group. Furthermore, in MD group moderate piloerection as well as the individual animal with abnormal breathing are assumed to be of toxicological relevance.

Body temperature of female HD animals was slightly increased at the end of the treatment.

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. A tendency to attenuation could be also mentioned for LD and MD groups during the gestation period.

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. Again, this was found as a tendency during the whole gestation period. For MD animals a tendency to decreased food consumption was also found during the gestation period.

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. The values were comparable between the groups. 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.

A reduced copulation index (when compared to C animals) was detected for LD, MD, and HD animals. Respective 90 % could be found. This is not assumed to be test item related. Fertility index, delivery index, as well as viability index were not different when compared to C animals.

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. Few incidences of external findings were observed in all groups (e.g. dark snout)which were considered to be spontaneous and not related to the test item.

At the end of the treatment period a slight tendency towards an increased amount of white blood cells could be observed in male HD animals (6.04 x 103/µl) when compared to C animals (4.68 x 103/µl).

In female animals, an increased percentage of monocytes could be observed. This increase was dose dependent but not statistical significant. 1.88 % (C ), 2.48 % (LD), 5.78 (MD), and 12.98 % (HD) were measured.

Percentage of eosinophils of female animals was significantly increased in MD animals (0.83 %) when compared to C animals (0.24 %). Furthermore, percentage of eosinophils in HD group was slightly but not significantly increased with 0.65 %).

At the end of the treatment period levels of Cholesterol were significantly increased in male LD group (1.17; p<0.05) and HD group (1.18; p<0.05) when compared with C group (0.86).

Levels of TBA were significantly increased (p<0.05) in male LD group when compared with C group. 20.26 as well as 9.3 could be measured, respectively.

In female animals, values of SGOT (ASAT) were slightly (p>0.05) decreased in treatment groups. 47.0 (C ), 38.7 (LD), 39.1 (MD), as well as 35.2 (HD) were measured. These slight decreases were not statistically significant and not dose dependent.

Values of Phosphate were slightly decreased in HD group (1.74) when compared to C group (3.21). This decrease was not statistical significant.

The urinalysis performed in male animals revealed no test- item related effect in any of the treatment groups compared to the control group. The urine analysis in female animals revealed increased blood content in HD animals as well as an increased protein content in treatment groups. Since no systematic finding was detected during histopathology and clinical biochemistry and since no organ weight changes were measured a test item relation and toxicological relevance seems unlikely. Especially in case of the blood content, contaminations during the necropsy could be responsible for this. 

At the end of the treatment period, no influence of the test item could be found on sperm motility or sperm head counts.

Few specific gross pathological changes were recorded for the male and female animals of the HD group which were assumed to be test item related and of toxicological relevance.

In male animals absolute weight of spleens was slightly increased in MD groups and was significantly increased (p<0.05) in HD group. This could be partly confirmed by weights in relation to body weight. In female animals, absolute weights of spleens of HD group were slightly increased when compared to C group. This could be confirmed by relative spleen weights of female animals. A toxicological relevance seems unlikely but however cannot be excluded for the male HD group.

Absolute prostate weights (plus seminal vesicles and coagulating glands) was decreased in male HD group when compared to C group. This could be confirmed by relative weights (to body weight). Due to the high variability as well as the absent histopathological findings a test item relation is not assumed.

In male animals, absolute weights of thymi were slightly increased in HD group when compared to C group. Relative organ weights of male animals showed no alteration. In female animals, absolute weights of thymi were slightly decreased. This could be confirmed by relative thymus weights (to body weight). Due to the mild characteristics of the increase (female) and decrease (male) as well as the opposite pattern in male and female animals a test item relation cannot be clearly stated. A toxicological relevance within this study cannot be considered.

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. No directly test item-related histopathological findings were noted in the other organs evaluated in this study. 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.