Registration Dossier
Registration Dossier
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EC number: 205-011-6 | CAS number: 131-11-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
There is a OECD 422 screening study available for DMP. The NOAEL for general toxicity and for reproductive and developmental toxicity was the inclusion level of 15000 ppm (corresponding to 1007 mg/kg/day for males and 1595 mg/kg/day for females).
The OECD 422 study is utilized to facilitate read-across to DEP, a close structural analogue. DEP was tested in a 2-generation study by Fuji et al. [2005] in Sprague-Dawley rats via the feed. The NOAEL was set at the mid dose in F1 and F2 animals due to reduced body weight and food consumption at 1000mg/kg. No effects on fertility were observed. Additionally, DMP was tested in a rat uterotrophic assay [BASF AG, 1999] in female immature Wistar-rats and results for estrogenic activity were negative.
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Mar 2022 - Mar 2023
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- - Batch no.: WM_21818_1930
- Purity: 99.9 %
- Expiry date: 18/08/2022
- Appearance: Clear, colourless liquid
- Storage conditions: Room temperature
- The determination of the identity, strength, purity, composition and stability of the test item was the responsibility of the Sponsor. The Sponsor declared that the characterisation of the test item was carried out according to a GLP quality system. A certificate of analysis is available. - Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: (P) x 10 - 11 weeks old
- Weight at study initiation: (P) 328-335 g for males and 218-221 g for females
- Fasting period before study: no
- Housing: From arrival to mating, animals were housed up to 5 of one sex to a cage. During mating, animals were housed one male to one female per cage. After mating, the males were re-caged as they were before mating. The females were transferred to individual cages for the gestation period, birth and lactation.
- Diet: ad libitum.
- Water ad libitum.
- Acclimation period: 26 days.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C±2 °C
- Humidity (%): 55%±15%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day
IN-LIFE DATES: From: 15 March 2022 To: 22 May 2022 - Route of administration:
- oral: feed
- Vehicle:
- other: powdered rodent diet (4RF21 Mucedola s.r.l, Settimo Milanese (MI), Italy)
- Details on exposure:
- DIET PREPARATION
- The test item was formulated, using powdered diet, by initial preparation of a pre-mix followed by dilution with further quantities of diet and mixing. The formulations were prepared separately for each group. Fresh diets were prepared at weekly intervals, based on the stability data, unless specified otherwise, at fixed concentrations of 1500, 5000 and 15000 ppm. Concentrations were calculated and expressed in terms of test item as supplied.
VEHICLE
- Concentration in vehicle: 0, 1500, 5000, 15000 ppm - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: The female was paired with the same male until positive signs of copulation was observed.
- Proof of pregnancy: spermidentification, vaginal plug in situ or copulation plugs found in the cage tray, the day of successful mating referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individual - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- In order to validate the analytical method and the formulation procedure and to verify the stability of the preparations, an analysis was performed in a separate study . The 24-hour and 8-day stability at room temperature were verified in the range from 1500 to 15000 ppm. The proposed preparation procedure for the test item was checked in the range from 1500 to 15000 ppm by chemical analysis (concentration and homogeneity) to confirm that the method was suitable. Final results for all levels were within the acceptability limits for concentration (80-120%) and homogeneity (CV < 15%).
Samples of the preparations prepared on Week 1 and Last week were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits (80-120% for concentration and CV < 15% for homogeneity) with the exception of Group 2M/F in the last week (recovery of 63.17%). Considering that this event occurred during the last week of dosing, limited to the recovery and not to the homogeneity (CV value), it may not have influenced the study. Moreover, no clinical signs were noted in those treated groups (mid- and high dose groups) where the results of the analysis were within the acceptability limits. - Duration of treatment / exposure:
- males: 34 days
females: ≥ 51 days - Frequency of treatment:
- daily
- Dose / conc.:
- 0 ppm
- Dose / conc.:
- 1 500 ppm
- Remarks:
- males: 105 mg/kg/day
females: 156 mg/kg/day - Dose / conc.:
- 5 000 ppm
- Remarks:
- males: 353 mg/kg/day
females: 532 mg/kg/day - Dose / conc.:
- 15 000 ppm
- Remarks:
- males: 1007 mg/kg/day
females: 1595 mg/kg/day - No. of animals per sex per dose:
- 10 animals/sex/dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Dose levels were selected by the Sponsor based on information from a 2-weeks preliminary non-GLP compliant study. Based on the results obtained in this preliminary study, it can be concluded that the test substance, when mixed with powdered rodent diet at the inclusion levels of 5000 and 15000 ppm (corresponding to approximately 400 and 1200 mg/kg/day) in terms of test item as supplied, resulted to be palatable and well tolerated by Wistar Hannover rats.
- Fasting period before blood sampling for clinical biochemistry: yes - Positive control:
- Not applicable
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality, at least once daily for clinical signs
DETAILED CLINICAL OBSERVATIONS (Functional Observation Battery Tests): Yes
- Time schedule: Once before start of treatment and weekly thereafter
- 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, stereo- typies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
- Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded. All observations were recorded for individual animals. Animals were examined in an open arena for a minimum of three minutes.
BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7 and 13 post partum and just before to necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Males: Food consumption was recorded at weekly intervals (whenever possible) starting from the day of allocation to the day of dosing or the day before and weekly thereafter until to mating. Daily food consumption was recorded during the mating period. After the end of the pairing period, food consumption was measured in two occasions (Days 18 and 20 of the mating phase).
- Females: Food consumption was recorded at weekly intervals (whenever possible) starting from the day of allocation to the day of dosing or the day before and weekly thereafter until to mating. Food consumption was also recorded daily during post coitum period, starting from Day 0 post coitum up to the day of parturition and daily during post partum period starting from Day 0/1 post partum. During gestation period, food consumption will be reported in the report until Day 20 post coitum (both for individual and group mean data). For female n. 57 (Group 3) which was mating not detected, the food consumption was reported before mating and during lactation period.
- Achieved dosage: At weekly intervals the achieved intake of test item was calculated from the mean weekly body weight, food consumption data and the dietary inclusion levels of the test item, where possible.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: During the last week of treatment (males on Day 32 and females on Day 13 post partum)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 males and 5 females
- Parameters: 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: Prothrombin time, Activated partial thromboplastin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period, (males on Day 32 and females on Day 13 post partum); at termination for T4/TSH
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 males and 5 females; all animals for T4 and TSH
- Parameters: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride, Inorganic phosphorus; T4/TSH
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Grip strength and sensory reactivity to stimuli: Once during the study, towards the end of treatment (Day 12 post partum for females with viable litters), 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. For males, the tests were performed on Day 29 of the study. Measurements were performed using a computer generated random order.
- Motor activity assessment (MA): Once during the study, towards the end of treatment (on Day 12 post partum for females with viable litters), 5 males and 5 females were randomly selected from each group and the motor activity (MA) was measured (for approximately continuous 60 minutes) by an automated activity recording device. For males, the tests were performed on Day 29 of the study. Measurements were performed using a computer generated random order.
IMMUNOLOGY: No
OTHER:
MATING:
Pairing was monogamous (one male to one female). A daily vaginal smear was taken from all females 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). The female was paired with the same male until positive signs of copulation was observed.
PARTURITION AND GESTATION LENGTH:
- A parturition check was performed from Day 20 to Day 25 post coitum. A parturition check was performed three times a day during the working day and twice daily during the weekends and Public Holidays.
- Female which did not give birth after 25 days of post coitum period was sacrificed shortly after (Group 2 - no. 27 on Day 28 post coitum).
- Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth. The day of birth (when parturition was complete) was defined as Day 0 post partum. - Oestrous cyclicity (parental animals):
- VAGINAL SMEARS AND OESTRUS CYCLE:
- Vaginal smears were taken in the morning from Day 1 of dosing up to positive identification of mating. The vaginal smear data was examined to determine the following: 1. anomalies of the oestrous cycle; 2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
- Before despatch to necropsy: Vaginal smears were also taken from all females, before despatch to necropsy and the oestrous cycle phase recorded. - Sperm parameters (parental animals):
- Parameters examined in all P male parental animals:
- identification of the stages of the spermatogenic cycle in all control and high dose males - Litter observations:
- PUPS IDENTIFICATION, PUPS WEIGHTS AND OBSERVATIONS:
- As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
- Live pups were individually weighed on Days 1, 4, 7 and 13 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy.
- Observation was performed once daily for all litters from Day 0 post partum until termination. After culling, all pups were sacrificed with the dams on Day 14 post partum.
CULLUNG AND PUPS SELECTION FOR BLOOD COLLECTION (SERUM HORMONE DETERMINATION AT NECROPSY):
- On Day 4 post partum, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment (for example, 5 males and 3 females) was acceptable.
- On Day 4 post partum, at least one culled male and one culled female per litter were selected for hormone determination.
- No pups were eliminated when litter size dropped below the culling target (8 pups/litter). If there was only one pup available above the culling target, only one pup was eliminated and used for blood collection for possible hormone determination.
ANOGENITAL DISTANCE (AGD):
- The AGD of each pup was measured on Day 1 post partum. The AGD was normalized to the cube root of body weight collected on Day 1 post partum.
NIPPPLE COUNT:
- The presence of nipples/areolae in male pups was checked on Day 13 post partum.
NIPPLE RETENTION:
- The nipples/areolae in male pups were checked during the necropsy procedure. No nipples were present.
PUP ORGAN WEIGHT
- Pups at Day 14 post partum: Thyroid was weighed from one male and one female pup selected for blood collection and preserved in 10% neutral buffered formalin.
- The thyroid weight was determined after fixation.
THYROID HORMONES
- Samples from pups on Day 14 post partum
- Parameters: T4 and TSH - Postmortem examinations (parental animals):
- GROSS PATHOLOGY: Yes (see table 1)
HISTOPATHOLOGY: Yes (see table 1) - Postmortem examinations (offspring):
- PUPS NECROPSY:
- Pups: All pups found dead in the cage were examined for external and internal abnormalities.
- Pups at Day 4 post partum: All culled pups sacrificed at Day 4 post partum were subjected to an external examination.
- Pups at Day 14 post partum: All live pups sacrificed on Day 14 post partum were examined for external abnormalities.
- Internal examination. Gonads were inspected from all pups in order to confirm the sex previously determined by external examination. - 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.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the non- parametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if ‘n’ was more than 5. - Reproductive indices:
- Males:
Copulation Index (%) = no. of males with confirmed mating / no. of males cohabitated ×100
Fertility Index (%) = no. of males which induced pregnancy / no. of males cohabitated ×100
Females:
Copulatory Index (%) = no. of females with confirmed mating/ no. of females cohabitated ×100
Fertility Index (%) = no. of pregnant females /no. of females cohabitated ×100
Males and females:
Pre coital Interval = The number of nights paired prior to the detection of mating
Sex ratios was calculated at birth, on Day 4 and on Day 14 post partum and was presented as the percentage of males per litter. - Offspring viability indices:
- Pre-natal loss (%) = (no. of visible implantations−Live litter size at birth) / no. of visible implantations ×100
Post-natal loss, day 0 (%) = (Total litter size−Live litter size) / Total litter size ×100
Post-natal loss, day 4 before culling (%) = (Live litter size at birth−live litter size at Day 4 (before culling)) / Live litter size at birth ×100
Post-natal loss, day 13 (%) = (Live litter size on Day 4 (after culling)−Live litter size on Day 13) / Live litter size on Day 4 (after culling) ×100 - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment-related clinical signs occurred during the study.
- Mortality:
- no mortality observed
- Description (incidence):
- No mortality occurred throughout the study.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No relevant differences were found in body weight and body weight gain between control and treated males and females throughout the study.
The statistically significantly higher in body weight gain noted on Day 8 of the mating phase in males of Groups 3 and 4 (inclusion levels of 5000 and 15000 ppm) was considered not relevant. The negative gain weight noted in all male groups on the last measurement was due to the fact that the animals were placed under condition of food deprivation for blood collection. The statistically significantly lower body weight gain observed on Day 7 post coitum in females of Group 4 was considered incidental considering the single occurrence which was followed by regular growth. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No effects on food consumption were observed in either males or females during the treatment period.
The mean overall achieved dosages for males were 105, 353 and 1007 mg/kg/day for females and 156, 532 and 1595 mg/kg/day against a fixed inclusion level of 1500, 5000 and 15000 ppm of test item in the diet. - Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Haematology:
- No treatment-related changes were recorded. A statistically significant difference of large unstained cells was recorded between control and males dosed at 15000 ppm. Due to the absence of other related changes at haemato- logical analyses or histopathology examination, this finding was considered to be incidental.
Coagulation and Blood clotting time:
- No treatment-related changes of blood clotting time, prothrombin time or activated partial thromboplastin time were recorded. The statistically significantly higher of prothrombin time recorded in males dosed at 5000 ppm was not dose-related, therefore it was considered to be incidental. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related changes were recorded. Albumin/globulin ratio was statistically significantly lower than controls in males dosed at 15000 ppm. Since albumin and globulin showed no changes, this finding was considered of no biological relevance. The statistically significant difference of phosphorus recorded between control and treated females were within the range of historical control data and not clearly dose-related, therefore this difference was considered to be unrelated to treatment.
No changes were recorded in the determination of T4 and TSH performed on samples from all parental males, between control and treated groups. - Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment. Motor activity was statistically significantly higher in females at the high dose level. However, the values were in the expected range for the rat strain used.
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- No treatment-related changes were observed at histopathologic evaluation in treated animals, when compared to the controls.
There were no test item-related microscopic observations in the testis (staging of the spermatogenic cycle). - Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- Oestrous cycle, reproductive parameters, pairing combination and mating performance:
- The total number of oestrous cycles, pre-coital intervals, copulatory and fertility indexes did not show any differences between groups.
- The total number of oestrous cycles observed in all females before pairing (number of days between the females were in oestrous) was similar between control and treated groups and had a mean value of 3 times.
- Vaginal smears examined on the day of necropsy to determine the stage of the oestrous cycle showed the phase of dioestrous for the all females sacrificed on Day 14 post partum and for that sacrificed on gestation phase.
- Pre-coital interval values of the treated females were comparable to the control. The majority of females had a sperm positive identification between 1-5 days of cohabitation.
- The number of copulation plugs (3/4 as mean value) was similar between control and treated groups.
- Copulatory and fertility indices were unaffected by treatment with the test item. - Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- No treatment-related changes were observed at the staging of the spermatogenic cycle.
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- Parturation:
- One control female had unilateral implantation and one female of Group 2 was found not
pregnant at necropsy.
- The number of pregnant females was: 10, 9, 10 and 10 in Groups 1, 2, 3, and 4 respectively.
- The number of females with live pups on Day 14 post partum was 9, 9, 10 and 10 in Groups
1, 2, 3 and 4, respectively.
Implantation, pre-natal loss data and gestation length of females:
- Gestation periods were similar in treated groups and controls. All pregnant dams gave birth on Day 22 post coitum (mean value).
- Number of implantations sites, live litter size, pre-implantation and pre-natal losses of treated groups appeared comparable to control values.
- Pre-natal loss was higher in Group 2, but there was no dose dependency. - Dose descriptor:
- NOAEL
- Effect level:
- >= 1 009 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other: No adverse treatment-related effects observed up to the highest tested dose.
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment-related clinical signs were observed in pups during the 14-day observation period.
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- On Day 4 post partum, a higher increase in mean post-natal loss was observed in Group 4 females, compared to the control (2.78%, 1.76%, 2.60% and 6.34% in controls and ascending dose groups, respectively). In particular, the litter size of female nos. 69, 71 and 77 was slightly reduced, compared to Day 1 since pups were found dead or missing. However, the values are within the historical control range of the rat stain used (0 to 7.29). Therefore, this difference is not considered to be toxicologically relevant.Litter weight and mean pup weights were similar between control and treated groups, both on Days 4 and 7 post partum.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No treatment-related effects were observed.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No significant differences were observed in the weight of the thyroid in treated pups, when compared to controls.
- Sexual maturation:
- no effects observed
- Description (incidence and severity):
- No treatment-related differences were observed in sex ratio between treated and control litters at birth and on Days 1, 4 and 14 post partum.
- Anogenital distance (AGD):
- no effects observed
- Description (incidence and severity):
- No differences in the anogenital distance (normalised value) performed on Day 1 post partum, were noted between control and treated pups.
- Nipple retention in male pups:
- no effects observed
- Description (incidence and severity):
- No nipples were observed in male pups on the day of necropsy.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No findings were recorded at necropsy in pups found dead after birth. Autolysed process, normally occurs when pups are found dead some time after death, therefore it is not considered to be treatment-related. No significant findings were seen in pups killed on Days 4 and 14 post partum.
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- >= 1 009 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other: No adverse treatment-related effects observed up to the highest tested dose.
- Critical effects observed:
- no
- Reproductive effects observed:
- no
- Conclusions:
- Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and for reproductive and developmental toxicity was the inclusion level of 15000 ppm (corresponding to mean achieved dose levels over the entire period of treatment of 1009 mg/kg/day for males and 1595 mg/kg
- Executive summary:
The toxic effects on Wistar rats of both sexes after repeated dosing with the test substance by oral route, as well as, effects of the test item on male and female reproductive performance, were investigated in a GLP-conform study according to OECD 422. The test substance was administered via diet at 0, 1500, 5000 and 15000 ppm, corresponding to dosage levels of 105, 353 and 1007 mg/kg/day for males and 156, 532 and 1595 mg/kg/day for females.
The treatment period was up to 34 days for males, including 2 weeks prior to pairing, the pairing period and until the day before necropsy. The treatment period of females sacrificed at termination was for a minimum of 51 days for females and included the 2 week period prior to pairing, the pairing period, the gestation phase and post partum phase until Day 13.
No mortality occurred throughout the study. One control female had unilateral implantation and one female of Group 2 was found not pregnant at necropsy. The number of pregnant females was: 10, 9, 10 and 10 in Groups 1, 2, 3, and 4 respectively. The number of females with live pups on Day 14 post partum was 9, 9, 10 and 10 in Groups 1, 2, 3 and 4, respectively.
Regarding clinical signs, no treatment-related clinical signs occurred during the study.
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal any changes attributable to the test item.
No relevant differences were found in body weight and body weight gain between control and treated males and females throughout the study.
No effects on food consumption were observed in either males or females during the treatment period. The mean overall achieved dosages for males were 105, 353 and 1007 mg/kg/day for males and 156, 532 and 1595 mg/kg/day against a fixed inclusion level of 1500, 5000 and 15000 ppm of test item in the diet.
No treatment-related alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.
Regarding haematology, no changes were recorded. No treatment-related changes were recorded regarding Coagulation and Blood clotting time
No treatment-related changes were recorded in clinical chemistry parameters.
No treatment-related changes for T4 and TSH were recorded in parental males or in pups of both sexes at Day 14 post partum.
The total number of oestrous cycles, pre-coital intervals, copulatory and fertility indexes did not show any differences between groups.
Gestation periods were similar in treated groups and controls. All pregnant dams gave birth on Day 22 post coitum (mean value).
Number of implantations sites, live litter size, pre-implantation and pre-natal losses of treated groups appeared comparable to control values.
No relevant differences were observed in litter data between control and treated groups from the day of birth and Day 4 post partum. After culling litter weight and mean pups weight (for both sexes combined) of treated groups remained similar to control.
No treatment-related differences were observed in sex ratio between treated and control litters at birth and on Days 1, 4 and 14 post partum.
No treatment-related clinical signs of pubs were observed in pups during the 14-day observation period.
No differences in the anogenital distance (normalised value) performed on Day 1 post partum, were noted between control and treated pups.
No significant differences were observed in the weight of the thyroid in treated pups, when compared to controls.
Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not indicate treatment-related effect. No nipples were observed in male pups on the day of necropsy.
No treatment-related changes were observed in terminal body weight or absolute and relative organ weights of treated animals, when compared to the controls.
No treatment-related changes were observed at post mortem examination in treated animals, when compared to the controls.
No treatment-related changes were observed at histopathologic evaluation in treated animals, when compared to the controls.
There were no test item-related microscopic observations in the testis (staging of the spermatogenic cycle).
Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and for reproductive and developmental toxicity was determined to be 15000 ppm (corresponding to mean achieved dose levels over the entire period of treatment of 1009 mg/kg/day for males and 1595 mg/kg for females).
- Endpoint:
- two-generation reproductive toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: guideline study that meets basic scientific principles, well documented under conditions of GLP, study is sufficient for endpoint evaluation and reliable without restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
- Deviations:
- no
- GLP compliance:
- yes
- Specific details on test material used for the study:
- - Name of test material (as cited in study report): Diethylphthalate CAS-No.: 84-66-2
- Analytical purity: 99.8%, commercial grade
- Lot/batch No.: SEM5441; supplier: Wako Pure Chemical Industries Ltd., Tokyo Japan
- Stability under test conditions: tested, prepearations used within 44 days after preparation for which stability of test substance in the diet was confirmed by a preivous performed analysis, no further details - Species:
- rat
- Strain:
- other: Crj: CD(SD) IGS
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Tsukuba Breeding Center, Charles river Japan, Inc. , specific pathogen free animals at 4 weeks of age
- Age at study initiation: (P) 5 wks
- Housing: individual housing, except for acclimatisation period (one or two animals by sex), mating (1F/1M), and lactation periods (1 litter); in metallic bracket-type cages with wire mesh floors; form gestation day 17 to lactation day 21d (day of weaning), individual dams and litters were reared on bedding: Whiteflake, Charles River Japan Inc, Yokohama, Japan
- Diet: basal and diet feed ad libitum throughout the study; feed: NIH-07M, Clea Japan Inc., Tokyo Japan
- Water: ad libitum
- Acclimation period: yes, one week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/-3
- Humidity (%): 50 +/-20
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): test suibstance stable in feed for 44 days - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: housing in the males cage until sucessful copulation, daily observation of vaginal smears from week 9 of treatment and mating of animals was initated at week 11
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: yes (explain: preocesses were repeated within 3 weeks mating period until pregnancy results) - Duration of treatment / exposure:
- - Exposure period: 15 weeks for males and 17 weeks for females
- Premating exposure period (males): 10 weeks
- Premating exposure period (females): 10 weeks
- Duration of test: 15 weeks for males and 17 weeks for females; 2 generations - Frequency of treatment:
- daily via feed
- Details on study schedule:
- - F1 parental animals not mated until 27 days after selected from the F1 litters; F1 M and F were observed daily for preputial separartion from 35 days of age and for vaginal opening from 27 days of age to dtermine the day of completion, vaginal smears were GIEMSA staind and examined microscopically during mating period until signs of pregnancy were found
- Selection of parents from F1 generation when pups were 21-25 days of age. 96 healthy animals of each sex were selected at 5 weeks of age for use. The animals were weighed and assigned to 4 groups in such a way to equalize group means and standard deviations of body weights. Each group consisted of 24 males and 24 females, as F0 parental animals. All were assigned a unique number and ear tattooed, prior to initiation of the administration period. On PND 4, pups were selected by sex at random so that the number of pups in a litter was 8 (if possible, 4 males and 4 females), and the remaining pups were euthanized and autopsied. Weaning of pups was performed on PND 21. In each group, 24 males and 24 females were selected from F1 weanlings at 21 to 25 days of age to become F1 parental animals. The selection of F1 weanlings was performed from litters born during a 5-day period including the day of the largest number of parturitions among all the groups. The number of weanlings selected from each litter was 1 or 2 per sex and the mean body weights were equalized among groups as much as possible. F1 parental animals were reared for 10 weeks and bred to obtain F2 offspring in the same manner as described for FO parental animals. Sibling matings were not performed. The total length of administration to both FO and F1 parental animals was approximately 15 weeks for males and approximately 17 weeks for females. - Dose / conc.:
- 40 mg/kg bw/day
- Remarks:
- male, F0 generation, calculated mean DEP intake, 600 ppm nominal in diet, 28 - 64 mg/kg bw/day actual ingested
- Dose / conc.:
- 197 mg/kg bw/day
- Remarks:
- male, F0 generation, calculated mean DEP intake, 3000 ppm nominal in diet, 141 - 315 mg/kg bw/day actual ingested
- Dose / conc.:
- 1 016 mg/kg bw/day
- Remarks:
- male, F0 generation, calculated mean DEP intake, 15000 ppm nominal in diet, 721 - 1594 mg/kg bw/day actual ingested
- Dose / conc.:
- 51 mg/kg bw/day
- Remarks:
- female, F0 generation, calculated mean DEP intake, 600 ppm nominal in diet, 32 - 90 mg/kg bw/day actual ingested
- Dose / conc.:
- 255 mg/kg bw/day
- Remarks:
- female, F0 generation, calculated mean DEP intake, 3000 ppm nominal in diet, 160 - 453 mg/kg bw/day actual ingested
- Dose / conc.:
- 1 297 mg/kg bw/day
- Remarks:
- female, F0 generation, calculated mean DEP intake, 15000 ppm nominal in diet, 815 - 2191 mg/kg bw/day actual ingested
- Dose / conc.:
- 46 mg/kg bw/day
- Remarks:
- male, F1 generation, calculated mean DEP intake, 600 ppm nominal in diet, 29 - 73 mg/kg bw/day actual ingested
- Dose / conc.:
- 222 mg/kg bw/day
- Remarks:
- male, F1 generation, calculated mean DEP intake, 3000 ppm nominal in diet, 142 - 369 mg/kg bw/day actual ingested
- Dose / conc.:
- 1 150 mg/kg bw/day
- Remarks:
- male, F1 generation, calculated mean DEP intake, 15000 ppm nominal in diet, 722 - 1901 mg/kg bw/day actual ingested
- Dose / conc.:
- 56 mg/kg bw/day
- Remarks:
- female, F1 generation, calculated mean DEP intake, 600 ppm nominal in diet, 33 - 91 mg/kg bw/day actual ingested
- Dose / conc.:
- 267 mg/kg bw/day
- Remarks:
- female, F1 generation, calculated mean DEP intake, 3000 ppm nominal in diet, 158 - 428 mg/kg bw/day actual ingested
- Dose / conc.:
- 1 375 mg/kg bw/day
- Remarks:
- female, F1 generation, calculated mean DEP intake, 15000 ppm nominal in diet, 809 - 2140 mg/kg bw/day actual ingested
- No. of animals per sex per dose:
- 8
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- - Dose selection rationale: Dose levels were selected based on the results of a preliminary reproduction study in rats. In the preliminary study, Crj:CD (SD) IGS rats (as FO parental animals at 8 weeks of age), 8/sex/group, were given diets containing the test substance at dose levels of 0 (basal diet, control), 5000, 10000, 20000 or 40000 ppm for 4 weeks prior to mating and throughout the subsequent breeding period until weaning of F1 pups at 3 weeks of age. Clinical observation of parental animals revealed hematuria in 2 males at 20000 ppm and in 1 each of both sexes at 40000 ppm. Body weights of males and females were decreased throughout the experimental period at 40000 ppm. Relative liver weights were significantly increased in males at doses of 10000 ppm or more and absolute prostate weights were decreased at 20000 and 40000 ppm. Regarding the reproductive performance of parental animals, gestation length at 40000 ppm was reduced. Body weight gains of F1 pups were inhibited throughout the lactation period at 20000 and 40000 ppm and viability indices of F1 pups on lactation days 4 and 21 at 40000 ppm were remarkably decreased. Liver weights of male weanlings were decreased in all DEP-treated groups. Based on these results, 15000 ppm was selected for the high dose level in the present study because certain toxic effects on growth of the offspring would be expected. Then, the dose levels of 3000 and 600 ppm were selected for middle dose and low dose groups, respectively, dividing by a common ratio of 5.
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was examined twice daily for clinical signs and mortality throughout the experimental period.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was examined twice daily for clinical signs and mortality throughout the experimental period.
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights and food consumption were recorded weekly in both sexes before mating. In females after
copulation, body weights and food consumption of dams were recorded on GDs 0, 7, 14 and 20 and PNDs 0, 7, 14 and 21.
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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
OTHER:
REPRODUCTIVE PERFORMANCE
Only F1 animals were examined for sexual maturation. F1 males and females were observed daily for preputial separation from 35 days of age and for
vaginal opening from 27 days of age to determine the day of completion. Body weights on the day at completion were determined. Vaginal smears
were taken from each FO and F1 female, stained with Giemsa solution, and examined microscopically. The vaginal smears were checked for two weeks
before and during the mating period, until evidence of copulation was found. All FO and F1 females that copulated were allowed to deliver naturally. They were observed for signs of parturition at least three times (at approximately 9:00, 13:OO and 17:OO) daily from GDs 21 to 25. Females holding their pups under the abdomen to nurse them at 1:00 p.m. were considered to have completed parturition on that day. Cases where females had one or more live pups without any abnormalities such as dystocia were considered as normal deliveries. The gestation length was represented as the number of days from detection of copulation (GD 0) to completion of parturition. Pregnancy was confirmed by the occurrence of parturition or by the presence of implantation sites in the uterine horns at autopsy. The number of implantation sites in the uterus was counted for each female at autopsy.
Determination of cytochrome P450 isozyme contents in the liver:
Six F0 parental males of each group were selected for the determination of cytochrome P450. Microsomal fractions of the liver were prepared and the total amount of microsomal protein was determined according to a modification of the methods of Lowry (Lowry et al., 1951, Peterson, 1979). The contents of cytochrome P450 isozymes (CYPlA1/2, CYP2B1, CYP3A2 and CYP4A1) in the liver microsomes were determined by an immunochemical method using commercial primary antibodies (anti-rat polyclonal antibodies for each isozyme, Daiichi Pure Chemicals Co., Ltd., Tokai-mura, Japan). The analysis was performed at The Institute of Environmental Toxicology (Ibaraki, Japan).
Serum hormone levels:
The levels of serum testosterone and progesterone in F0 males were determined to investigate the effects on metabolism of these steroid hormones in the same animals for which P450 isozyme contents were determined. At autopsy, blood samples were collected from the abdominal aorta of these rats. Total testosterone and progesterone levels were measured with radioimmunoassay kits (Japan DPC Co., Chiba, Japan) at Panapharm Laboratories Co., Ltd. (Kumamoto, Japan). - Oestrous cyclicity (parental animals):
- not examined
- Sperm parameters (parental animals):
- Parameters examined in [all/P/F1] male parental generations:
- testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology,
- other: Sperm parameters were determined in all FO and F1 male adults, except for males found dead or killed during the study, on the day of the scheduled terminal sacrifice. The right cauda epididymis was weighed and used for sperm analysis. The right testis was also employed, for counting homogenization-resistant testicular spermatids. The cauda epididymis was placed in a petri dish containing 4 mL of medium (Medium 199, GIBUCO, Gland Island, NY, USA) supplemented with bovine serum albumin (0.5%) (Cohn Fraction V Powder, INTERGEN, NY, USA), cut into 6 pieces with a surgical knife, and incubated in a C02 incubator at 37°C and 5% CO2 for 5 min. After incubation, 0.05 mL of the epididymal fluid was put into a 1.5 mL capillary tube containing 0.45 mL of the medium and incubated in the C02 incubator for 5 min to diffuse the sperm. Sperm motility was analyzed using a computerassisted cell motion analyzer (HTM-IVOS, Hamilton Thorne Research, Beverly, MA, USA) and the percentage of motile sperm and progressively motile sperm, as well as the swimming speed and pattern, were determined. After recording sperm motion, cauda epididymal fluid was diluted and the sperm were enumerated using a hematocytometer under a light microscope. After being weighed, the tunica albuginea was removed from the testis and the parenchyma was homogenized in phosphate buffer solution (PBS), then sperm heads were also enumerated, again using a hematocytometer under a light microscope. Sperm counts were expressed as the number per cauda epididymis or testis and per gram cauda epididymis or testis. The sperm were stained with eosin and smeared on a slide glass and two hundred in each sample were examined under a light microscope, to determine the percentage which were morphologically abnormal. - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
- The following parameters were examined in [F1 / F2] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:]
GROSS EXAMINATION OF DEAD PUPS:
- yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.
- All F1 and F2 pups were examined for clinical signs and mortality daily before weaning. For each litter delivered normally, each pup was sexed externally and the numbers of males, females and the total were recorded on PND 0. In each litter, pups were individually weighed on PNDs 0, 4, 7, 14 and 21. Anogenital distance (AGD) was measured using a caliper on PNDs 0 and 4 for all F 1 and F2 pups. The number of pups surviving in each litter was counted on PNDs 0, 4 and 21 and viability indices were calculated.
OTHER
Physical development and reflex ontogeny:
All F1 and F2 pups were observed daily for pinna detachment on PNDs 1 to 4, incisor eruption from PND 8 to the day of completion, and eye opening from PND 12 to the day of completion. One male and one female of the F1 and F2 pups selected from each litter were evaluated daily for surface righting reflex from PND 4, negative geotaxis reflex from PND 7, and midair righting reflex from PND 13, each until the day of completion. - Postmortem examinations (parental animals):
- SACRIFICE
- Male/Maternal animals: All surviving animals:All F0 and F1 parental animals were euthanized by exsanguination under ether anesthesia and autopsied after weaning of their F1 and F2 offspring. The animals that failed to produce offspring were also euthanized by exsanguination under ether anesthesia and autopsied.
- other: In the control and high-dose groups, all F0 and F1 parental animals were examined histopathologically for abnormalities of the reproductive organs (i.e., ovaries, uterus, and vagina, or testes, epididymides, seminal vesicles, coagulating glands, and prostate), and of the pituitary gland, thyroid glands, liver, adrenal glands, and mammary glands. In the low dose group, histopathological examination of the same organs as the control and high-dose groups was also performed only for females that showed abnormal estrous cyclicity, males that showed sperm abnormality, and pairs of animals that failed to mate or produce offspring. In addition, the kidneys of F1 parental female animals in the control and high-dose groups were examined histopathologically, because a significant increase in the organ weight was noted in F1 parental females of the high-dose group as compared with controls. In F1 and F2 weanlings, histopathological examination was performed for the thymus of six each of the F1 and F2 males and females, and for the spleen of six F2 males each in the control and 15000 ppm groups, because the thymus weights in both sexes of the F1 and F2 weanlings and the spleen weights of F2 male weanlings were significantly decreased at 15000 ppm as compared with the control values.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGHTS
Major organs were fixed and preserved in neutral, phosphate-buffered 10% formalin. The testes and epididymides were fixed with Bouin's solution and preserved in 70% ethanol. Weights of the brain, liver, kidneys, spleen, pituitary gland, adrenal glands, testes, epididymides, prostate (ventral lobe), ovaries and uterus were recorded before fixation. Weights of the thyroid glands and seminal vesicles (with coagulating glands) were measured after fixation. For the bilateral organs, values were the sums of the weights for both sides.
- Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 26 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: F1 and F2 pups not selected on PND 4 were euthanized by inhalation of carbon dioxide and autopsied on that day. F1 weanlings not selected as F1 parental animals and all F2 weanlings were euthanized and autopsied according to the same methods as for the parental animals, at 26 days of age. The same organs as the parental animals and the thymus of one pup per sex, which was selected at random from each litter of the F1 and F2 weanlings in each group, were weighed. The same organs and tissues as their parental animals were fixed and preserved.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated above were prepared for microscopic examination and weighed, respectively.
- Statistics:
- Body weights, body weight gain, food consumption, length of the estrous cycle, gestation length, numbers of implants and pups delivered, the delivery index, sperm parameters, P450 isozyme contents, hormone levels, organ weights, organ/body weight ratios (relative organ weights), age completed reflex responses, pinna detachment, incisor eruption and eye opening, age and body weights at sexual maturation, AGD, and viability of pups were analyzed for statistical significance in the following way: homogeneity of variance was evaluated first by Bartlett's test (p<=0.05). When group variances were homogeneous, the one-way analysis of variance (p<=O.lO) was used to determine if any statistical differences existed among the groups. If the analysis of variance gave a significant result, Dunnett's test (p<=0.05 or 0.01) was performed to detect any significant differences between the treated groups and their corresponding controls. When Bartlett's test indicated that the variances were not homogeneous, the Kruskal-Wallis test (p<=O.10) was used for detecting any statistical differences and if they were significant, the Mann-Whitney U test (p<=0.05 or 0.01) was performed to detect any significant differences between the treated groups and their corresponding controls (Gad, 2001). Statistical analyses were performed using the litter averages for body weights and the AGDs of pups per sex before weaning. The Wilcoxon rank-sum test (p<=0.05 or 0.01) was employed to analyze the data on completion of physical development of pups using the litter as the unit for statistical evaluation (Gad, 2001). The incidences of females with normal estrous cyclicity, and indices of copulation, fertility and gestation, and sex ratios of pups were analyzed by the Chisquare test or Fisher's exact probability test (p<=0.05 or 0.01) (Gad, 2001).
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- effects observed, treatment-related
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Dose descriptor:
- NOAEL
- Effect level:
- >= 15 000 ppm
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects at the highest tested dose
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Sexual maturation:
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- no effects observed
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 3 000 ppm
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects at this dose
- Dose descriptor:
- LOAEL
- Generation:
- F1
- Effect level:
- 15 000 ppm (nominal)
- Sex:
- male/female
- Basis for effect level:
- sexual maturation
- body weight and weight gain
- Dose descriptor:
- NOAEL
- Generation:
- F2
- Effect level:
- 3 000 ppm
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects at this dose
- Dose descriptor:
- LOAEL
- Generation:
- F2
- Effect level:
- 15 000 ppm
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Reproductive effects observed:
- not specified
- Executive summary:
A two generation reproduction toxicity study was performed in Sprague-Dawley rats via the feed to evaluate the effects of diethyl phthalate on parental reproduction performance, including features of the endocrine system and development and growth of the offspring at dietary dose levels of 0, 600, 3000 and 15000 ppm (nominal concentration in diet). In F0 and F1 parents, no treatment related adverse effects were observed in clinical findings, body weights, food consumption, reproductive parameters and gross- or histopathological findings in any treated group. Increased liver weights and enhanced activities of metabolic enzymes were observed in F0 males at 15000 ppm. F0 males also exhibited an increase in the content of CYP3A2, a cytochrome P450 isoenzyme, at 15000 ppm, and a decrease in the levels of serum testosterone at 3000 and 15000 ppm, suggesting sex steroid metabolism might be changed. However, these were not considered adverse effects because the degree of change was to slight to affect the reproductive capability to produce progeny. Body weight gains before weaning were inhibited in F1 and F2 pups and vaginal opening was slightly delayed in F1 females at 15000 ppm. No changes were observed in the reproductive performance.
The NOAEL from this study is considered 15000 ppm for parental animals and 3000 ppm for development and growth of pups.
Referenceopen allclose all
Table. 2: Summary details of the pregnant status (P0)
Groups (ppm) | 1 (0) | 2 (1500) | 3 (5000) | 4 (15000) |
Initial group size | 10 | 10 | 10 | 10 |
Non-pregnant females | 0 | 1 | 0 | 0 |
Total litter loss | 1 | 0 | 0 | 0 |
Unilateral implantation | 1 | 0 | 0 | 0 |
Mating not detected, pregnant | 0 | 0 | 1 | 0 |
Number of pregnant females | 10 | 9 | 10 | 10 |
No. of females at term (with live pups on Day 14 post partum) | 9 | 9 | 10 | 10 |
Table 3: Achieved dosage, males (P0)
Group | Inclusion level (ppm) | Achieved dosage (mg/kg/day) | Study achieved dose (mg/kg/day) | |
Before pairing | Mating | Mean | ||
2 | 1500 | 106 | 104 | 105 |
3 | 5000 | 343 | 363 | 353 |
4 | 15000 | 998 | 1015 | 1007 |
Table 4: Achieved dosage, females (P0)
Group | Inclusion level (ppm) | Achieved dosage (mg/kg/day) | Study achieved dose (mg/kg/day) | ||
Before pairing | post coitum | post partum | Mean | ||
2 | 1500 | 121 | 135 | 214 | 156 |
3 | 5000 | 427 | 440 | 730 | 532 |
4 | 15000 | 1152 | 1340 | 2295 | 1595 |
- no compound-related clinical signs of toxicity in either parental males or females in either generation during the premating, mating, gestation or lactation periods.
- other: One F0 male in the 3000 ppm group was euthanized because of a nasal bone fracture due to an accident in the cage. One F0 female died of dystocia and another F0 female was euthanized because of pyometra in the control group.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- There were significantly increased body weights and body weight gain in FO females during the premating period in the 600 ppm group and a significantly increased body weight gain at treatment week 0-1 in F1 males in the 15000 ppm group (the value was 51.5 g as against 45.8 g in the control group). With FO females in the 15000 ppm group, body weight gain during lactation was significantly increased. These changes were not observed in F1 females. No significant differences were observed in any other treatment group.
- Significant reduction of food consumption was observed from the end of the premating period to gestation period in FO females in the 3000 ppm group. In the 15000 ppm group, there was a significant increase in both sexes of the F1 animals at treatment week 1, while no changes were observed in either sex of the FO animals.
TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- see body weight and food consumption for details
ORGAN WEIGHTS (PARENTAL ANIMALS)
- In the 15000 ppm group, absolute and/or relative liver weights in both sexes of the FO and F1 parental animals were significantly increased. In addition, absolute adrenal gland and epididymis weights in FO males were significantly decreased, with significantly low relative adrenal gland weights in F1 males and significantly high values for absolute and relative kidney weights in F1 females of this group.
OTHER:
- gestation length was significantly shortened in F1 females in the 15000 ppm group by 0.3 days
- significant increase in CYP4A1 and CYP3A2 were observed limited to the 15000 ppm dose group
- significant decrease in testosterone levels in the 3000 and 15000 ppm dose group in F0 males
- bw was significantly reduced in the 15000 ppm group on PND4 through 21 in F1 female pups, but there were no significant differences in bw at completion of vaginal opening any more
- In the 600 ppm group, no significant changes were observed except for a reduction of relative liver weights in F1 males. This change was considered incidental because of the lack of any dose-response relationship. In the 3000 ppm group, absolute adrenal gland weights in F1 females and relative uterine weights in F2 females were significantly decreased. These changes were also observed in the 15000 ppm group. In the 15000 ppm group, relative liver weights were significantly increased in both sexes of the F1 and F2 weanlings. In this group, absolute and relative thymus weights in both sexes of the F1 and F2 weanlings, absolute spleen weights in F1 males, and absolute and relative spleen weights in F2 males were significantly decreased. However, no histopathological abnormalities were observed in these organs. In addition, significant decreases were observed in absolute adrenal gland weights in both sexes of the F1 and F2 weanlings, in absolute prostate weights in F1 males, in absolute uterine weights in F1 females, and in absolute and relative uterine weights in F2 females in the 15000 ppm group. Changes in weights of the brain, kidneys, thyroid glands, pituitary gland and seminal vesicles in F1 and F2 weanlings were also observed.
SEXUAL MATURATION (OFFSPRING)
- age at completed vaginal opening was significantly delayed in in F1 females in the 15000 ppm group
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 222 mg/kg bw/day
- Study duration:
- chronic
- Species:
- rat
- Quality of whole database:
- Justification for NOAEL read-across: The potential human health hazard of the two phthalates (target and source substance) originates from the structural component of esterified phthalic acid present in both molecules. Moreover, both substances share toxicokinetic processes. Details can be found in the attached read across justification.
Additional information
The toxicity effects of Palatinol M, when administered by oral route (via diet) at inclusion levels of 1500, 5000 and 15000 ppm, to male and female Wistar Hannover rats was investigated in a OECD 422 guideline study (BASF 2023), as well as any possible effects on the reproductive performance (such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and early lactation of the offspring).
No mortality occurred throughout the study and no treatment-related clinical signs were noted during the study. No signs of neurotoxicity were observed during the study in parental males and females. No relevant differences in body weight and food consumption were observed in treated animals, compared to the control group. No treatment-related changes were observed in haematological or clinical chemistry parameters. Thyroid hormone evaluation in male parental animals, as well as in pups at Day 14 post partum did not show treatment-related changes.
The number of females with live pups on Day 14 post partum was 9 in the control group, 9 at 1500 ppm, 10 at 5000 ppm and 10 at 15000 ppm, since one female in Group 2 was found not pregnant and one in control group lost its litter during the first part of lactation period. No treatment-related anomalies were noted in the total number of oestrous cycles of the treated females, when compared to controls. Copulatory and fertility indices did not show any treatment related differences among treated and control groups. Parturition, lactation, implantation, litter data and sex ratio did not show changes. No differences in the anogenital distance (normalised value) were seen between control and treated groups both for male and female pups. Retained nipples were not detected in male pups. Necropsy findings and thyroid weight in pups did not reveal any treatment-related effect. No treatment-related changes were observed in terminal body weight or absolute and relative organ weights of treated animals of both sexes, when compared to the controls. No treatment-related changes were observed at post mortem macroscopic observations and microscopic evaluation, including the staging of the spermatogenic cycle. No treatment-related changes were observed in reproductive and developmental parameters.
Based on the results of the present study, the NOAEL for general toxicity and for reproductive and developmental toxicity was the inclusion level of 15000 ppm (corresponding to mean achieved dose levels over the entire period of treatment of 1007 mg/kg/day for males and 1595 mg/kg/day for females).
The OECD 422 study mentioned above can be utilized to facilitate read-across to DEP, a close structural analogue. DEP was tested in a 2-generation study by Fuji et al. (2005). The 2-generation reproduction toxicity study according to OECD TG 416 was performed to evaluate the effects of diethyl phthalate on parental reproduction performance, including features of the endocrine system and development and growth of the offspring at dietary dose levels of 0, 600, 3000 and 15000 ppm (nominal concentration in diet). Actual ingested doses in F0 males were: 28-64, 141-315, 721-1594 [mg/kg bw/day ], in F0 females: 32-90, 160-453, 815-2191 [mg/kg bw/day]; in F1 males: 29-73, 142-369, 722-1901 [mg/kg bw/day], in F1 females: 33-91, 158-428, 809-2140 [mg/kg bw/day]. In F0 and F1 parents, no treatment-related adverse effects were observed considering clinical findings, body weights, food consumption, reproductive parameters and gross- or histopathological findings in any treated group. Increased liver weights and enhanced activities of metabolic enzymes were observed in F0 males at 15000 ppm. F0 males also exhibited an increase in the content of CYP3A2, a cytochrome P450 isoenzyme, at 15000 ppm, and a decrease in the levels of serum testosterone at 3000 and 15000 ppm, suggesting sex steroid metabolism might be changed. However, these effects were not considered as adverse effects because the degree of change was to slight to affect the reproductive capability to produce progeny. Adverse effects are reduced body weight gains (F1: M -18%, F -19%; M -12%, F2 -12%; all values are in percentage compared to the control) before weanling in F1 and F2 pups. Furthermore, vaginal opening was slightly delayed in F1 females at 15000 ppm. Additionally, at the highest dose liver weights were increased in both male and female pups. However, no changes were observed in the reproductive performance. Therefore, the NOAEL from this study is considered to be 15000 ppm (nominal concentration, F0 and F1 parental toxicity) for parental animals and 3000 ppm for development and growth of pubs (nominal concentration, F1 and F2 offspring toxicity).
DMP was tested in a BASF AG [1999] uterotrophic assay similar to OECD TG 440. The test substance was applied orally (gavage) daily on 4 consecutive days to 10 immature female Wistar rats at two doses, 180.3 and 2008 mg/kg bw/day (analytical concentration). A standard dose volume of 5 ml/kg bw was used. The negative control (vehicle: olive oil) and the positive control DES-DP (diethylstilbestrol-dipropionate; 5 mg/kg bw/day) were both valid. DES-DP caused as expected an in crease in uterus weight. No substance related effects were detected for DMP. No mortalities occurred and no abnormalities were reported. Only one animal showed piloerection for 1 day (1 day after treatment), the following days were without abnormality. The test substance had no effect on uterine weight (absolute and relative). Thus, no estrogenic activity was observed.
Estrogen receptor binding was also assessed in vitro in several publications. DMP was not estrogenic in T47 -D human breast cancer cells (Legler 2002) or in two yeast reporter gene assay (Nishishara 2000, Harris 1997), nor did the test substance bind to estrogen receptors obtained from rat uterus cytosol (Murk 2000, Blair 2000).
No antiandrogenic or androgenic effects were observed in vivo (Gray 2000). Further details on this study are provided in the section on teratogenicity. Oishi (1980) fed diets containing 2% DMP (app. 2000mg/kg) to 10 male rats for one week. Tests and kidney weights as well as testes, liver, and kidney zink content were unaffected by treatment. There were no histopathological changes in the three examined organs. Liver weights were increased likely due to increased metabolism at this rather high dose. Decreased testosterone levels are considered secondary to increased metabolism, especially in combination with the negative results from the in vivo study by Gray (2000). Foster (1980) also did not observe changes in zink content of testes, liver and kidneys and no histopathological changes after treating rats with 1400mg/kg for four days.
In utero exposure to certain phthalate esters results in testicular toxicity, characterized at the tissue level by induction of multinucleated germ cells (MNGs) in rat, mouse, and human fetal testis. Spade (2018) exposed timed pregnant Sprague Dawley rats by daily oral gavage from gestation day 17 to 21 with one of eight phthalate test compounds or corn oil vehicle and counted MNGs. Both the manual counting method and the automated image analysis method identified di-nbutyl
phthalate, butyl benzyl phthalate, dipentyl phthalate, and di-(2-ethylhexyl) phthalate as positive for induction of MNGs. Dimethyl phthalate, diethyl phthalate, the brominated phthalate di-(2-ethylhexyl) tetrabromophthalate, and dioctyl terephthalate were negative. Furthermore DMP did not decrease testosterone ex vivo.
In summary, DMP does neither cause androgenic, antiandrogenic nor estrogenic effects in vivo and in vitro.
Effects on developmental toxicity
Description of key information
No teratogenic effects in rats in two studies according to or similar to OECD 414 (NTP, 1986; Hansen, 1989)
No effects on number of live / dead offspring in mice and rats (Plasterer, 1985; Hardin, 1987; Peters, 1973; Spade 2018)
No antiandrogenic or androgenic effects (Gray, 2000; Spade, 2018)
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- The study is reliable with restrictions and was conducted aquivalent or similar to OECD TG 414 (Prenatal development toxicity study). Restriction: the study was performed according to guideline recommendations using exposure from GD 5-16, but the critical time-window for phthalate developmental toxicity is from GD 16-18.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- - Name of test material (as cited in study report): Dimethyl phthalate (DMP)
- Physical state: clear, colorless liquid
- Analytical purity: >99% (gas chromatography)
- Lot/batch No.: 2113CM
- Stability under test conditions: yes, analyzed by the Research Triangle Institute supported by NTP
- Storage condition of test material: in amber bottles fitted with teflon-lined screw caps at -20°C
- other: DMP manufactured by the Aldrich Chemical Company and supplied by the Radian Corporation - Species:
- rat
- Strain:
- other: Crl:CD (SD)Br VAF/Plus outbred Sprague-Dawley rats
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Caesarean-originated, barrier sustained outbred Crl:SD (CD)BR VAF-Plus albino rats; supplier: Charles River Laboratories Inc., Raleigh, NC
- Age at study initiation: 8-12 weeks
- Weight at study initiation: not reported
- Fasting period before study: not reported
- Housing: random, M (single) and F (max. 3 per cage) in solid bottom polycarbonate cages (Laboratory Products, Rochelle Park, NJ)
- Diet: Purina Certified Rodent Chow, #5002, pelltized (for M breeders) or ground (F at beginning, switch to ground chow during cohabitation)
- Water: ad libitum (deionized, filtered water)
- Acclimation period: 7 days quarantine period prior to study
ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean 22.1-22.3
- Humidity (%): mean 54+-4
- Air changes (per hr): 12-14
- Photoperiod (hrs dark / hrs light): 12/12 (7.00p.m./7.00a.m.) - Route of administration:
- oral: feed
- Vehicle:
- other: food
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- DMP was weighed and added to an aliquot of Purina Certified Ground Rodent chowe (#5002) in a beaker and mixed with a spatula; premixed sample was thoroughly stirred by hand and it was added to the preweighed sample of ground feed from which the initial aliquot was taken and blended in a Patterson-Kelley Liquid-Solids Twin Shell Blender.
DIET PREPARATION
- Rate of preparation of diet (frequency): because of the limited stability of DMP under animal feeding conditions, animals were given a fresh supply of feed every third day throughout the dosing period.
- Mixing appropriate amounts with (Type of food): preweight ground food
- Storage temperature of food: formulated DMP/feed mixtures were refrigerated in light-protected containers - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - All feed formulations administered to the animals assayed within 90-110% of the theoretical concentration with the exception of the high dose (5.0%) in the preliminary study which was measured at 88.34%
- HPLC analysis - Details on mating procedure:
- - Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight in the cages of the single housed M
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0 of pregnancy - Duration of treatment / exposure:
- days 6-15 of gestation
- Frequency of treatment:
- continuous
- Dose / conc.:
- 200 mg/kg bw/day (actual dose received)
- Remarks:
- calculated based on average food intake and a dietary concentration of 0.25 % (nominal concentration)
- Dose / conc.:
- 840 mg/kg bw/day (actual dose received)
- Remarks:
- calculated based on average food intake and a dietary concentration of 1.0 % (nominal concentration)
- Dose / conc.:
- 3 570 mg/kg bw/day (actual dose received)
- Remarks:
- calculated based on average food intake and a dietary concentration of 5.0 % (nominal concentration)
- No. of animals per sex per dose:
- 14-15 sperm-positve F per treatment in replicates
- Control animals:
- yes, concurrent no treatment
- yes, historical
- Details on study design:
- Duration of test: gestation days 6-15
- Dose selection rational: dose selection after range finding study; highest dose was expected to cause limited adverse effect in the dam - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: GD 3,6-9, 9-12, 18-20
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
BODY WEIGHT: Yes
- Time schedule for examinations: GD 3,6,9,12,15,18,20
WATER CONSUMPTION : Yes
- Time schedule for examinations: 0-3, 3-6, 6-9, 9-12, 12-15, 15-18. 18-20
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterine, liver, kidney - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Middle resorption: Yes (some fetal tissue, in addition to early resorptions) - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter - Statistics:
- ANOVA; Williams and Dunnett; Multiple Comparison Test, Fishers exact probability test
- Historical control data:
- Yes, historical control data in the appendix of the final report (teratologic investigations in the CD rat at Research Triangle Institute)
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
see table 1+2 (remarks on results including tables and figures) - Dose descriptor:
- NOAEL
- Effect level:
- 840 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: No adverse effects at this dose
- Dose descriptor:
- LOAEL
- Effect level:
- 3 570 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- organ weights and organ / body weight ratios
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
see table 1+2 (remarks on results including tables and figures) - Dose descriptor:
- NOAEL
- Effect level:
- >= 3 570 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effects at the highest tested dose
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Conclusions:
- The NOAEL for maternal toxicity was 1.0% (840 mg/kg bw/day) DMP.
The NOAEL for developmental toxicity was 5.0% (3570 mg/kg bw/day) DMP. - Executive summary:
The study is reliable with restrictions and was conducted equivalent or similar to OECD TG 414 (Prenatal development toxicity study). Dimethyl phthalate (DMP) was evaluated for developmental toxicity in timed-pregnant Sprague-Dawley (CD) rats. On gestational days (GD) 6 through 15, dietary concentrations of 0.0, 0.25, 1.0 and 5.0% (nominal concentration) DMP were administered, resulting in an estimated average food intake of 0.0, 0.2, 0.8 and 3.6 g/kg bw/day. This study was conducted to clarify the conflicting situation in the literature. Singh et al. (1972) reported embryo lethality and malformations in the rat after DMP exposure whereas Plasterer et al. (1985) reported no substance related effects in the mouse. This study was conducted in the rat to check rat specific developmental toxicity; additionally the positive study by Singh et al. (1972) was not assignable due to small sample size and limited fetal examinations. However, the study was performed according to guideline recommendations using exposure from GD 5-16, but the critical time-window for phthalate developmental toxicity is from GD 16-18.
High-dose DMP resulted in transient reductions in food and water consumption and body weight during early treatment. When treatment ended, food and water consumption rose above controls. Animals fed 5.0% DMP also exhibited reduced body weight gain during the treatment period and increased relative liver weight. Neither 0.25% nor 1.0% DMP treatment had significant maternal effects, with the exception of reduced water consumption during early treatment and increased food and water consumption following treatment (1.0% only). These results suggest that the apparent toxic effects of high-dose DMP on body weight may reflect the unpalatability of DMP in feed.
DMP treatment during organogenesis produced no apparent effects on measured endpoints of embryo/fetal viability, growth or development. The implantation sites and percentages of resorptions, dead fetuses, nonlife implants or malformed fetuses per litter were similar among dose groups. DMP treatment had no effect on the number of live fetuses per litter or average fetal body weight per litter. There was no effect of treatment on the incidence of external, skeletal or visceral malformations.
In summary, in this study in CD rats, maternal toxicity was observed at a dietary treatment level of 5.0% DMP. No developmental endpoints were affected under the conditions of this study.The NOAEL for maternal toxicity was 1.0% (840 mg/kg bw/day) DMP.
The NOAEL for developmental toxicity was 5.0% (3570 mg/kg bw/day) DMP.
Reference
Table1: responses to DMP exposure
DMP in food (g/kg bw/day) |
||||
0.0 |
0.2 |
0.84 |
3.57 |
|
MATERNAL |
||||
Maternal body weight gd 9 + 12 |
-- |
-- |
-- |
↓ |
BW gain (treatment) |
-- |
-- |
-- |
↓ |
Relative liver weight |
-- |
-- |
-- |
↑ |
Food consumption (g/kg bw/day) gd 6-9 + 9-12 gd 15-18 |
-- -- |
-- -- |
-- -- |
↓ ↑ |
Water consumption (g/kg bw/day) gd 6-9 + 9-12 gd 18-20 |
-- -- |
-- -- |
-- -- |
↓ ↑ |
Embryo/fetal |
||||
% nonlive implants/litter |
-- |
-- |
-- |
-- |
% malformed fetuses/litter |
-- |
-- |
-- |
-- |
Average fetal bw/litter |
-- |
-- |
-- |
-- |
-- indicates no significant difference (p= 0.05) difference from control value
↓ or ↑ indicates significant difference (p= 0.05) difference from control value
Table2: Raw data from Fields et al. (1989) all data in (g) if not indicated different
DMP in food (g/kg bw/day) |
|||||
0.0 |
0.2 |
0.84 |
3.57 |
||
DMP % in feed |
0 |
0.25 |
1.0 |
5.0 |
|
MATERNAL TOXICITY |
|||||
Animals treated |
30 |
30 |
29 |
30 |
|
No. Dead |
0 |
0 |
0 |
0 |
|
No. (%) pregnant at sacrifice |
26(90) |
25(89) |
26(90) |
28(93) |
|
Maternal bw GD0 |
234.98+-2.75 |
236.25+-3.05 |
232.68+-2.30 |
239.96+-2.47 |
|
Maternal bw GD at sacrifice |
378.72+-5.17 |
386.69+-6.68 |
381.25+-4.58 |
378.12+-5.82 |
|
Maternal weight gain (gestation) |
143.74+-3.32 |
150.44+-4.54 |
148.57+-3.20 |
138.16+-4.68 |
|
Maternal weight gain (corrected for gravid uterine weight) |
64.38+-1.58 |
67.36+-3.02 |
67.07+-2.34 |
58.97+-2.34 |
|
Gravid uterine weight |
79.36+-3.20 |
83.08+-2.26 |
81.50+-3.24 |
79.19+-3.79 |
|
Maternal liver weight |
61.40+-0.23 |
17.03+-0.41 |
16.76+-0.35 |
17.33+-0.34 |
|
Maternal relative liver weight (% bw) |
4.31+-0.06 |
4.40+-0.06 |
4.39+-0.06 |
4.59+-0.06** |
|
Maternal right kidney weight |
1.153+-0.020 |
1.149+-0.026 |
1.114+-0.018 |
1.174+-0.024 |
|
Maternal left kidney weight |
1.112+-0.016 |
1.086+-0.040 |
1.077+-0.017 |
1.149+-0.020 |
|
Maternal food consumption (g/kg bw/day) 0-3 3-6 6-9 9-12 12-15 15-18 18-20 0-20 |
|||||
88.3+-3.1 |
91.9+-2.9 |
86.1+-2.1 |
83.6+-2.1 |
||
88.0+-1.7 |
92.8+-2.3 |
87.5+-2.4 |
95.4+-3.9 |
||
83.0+-1.2 |
79.4+-1.7 |
82.9+-2.5 |
59.5+-3.2** |
||
81.5+-3.5 |
82.6+-2.2 |
83.8+-1.5 |
70.1+-2.8** |
||
80.7+-1.6 |
81.1+-1.7 |
87.0+-2.8 |
85.1+-2.0 |
||
73.3+-1.3 |
79.8+-2.1 |
85.2+-2.2** |
90.8+-2.0** |
||
67.1+-2.4 |
68.5+-3.6 |
71.8+-3.2 |
76.5+-2.9 |
||
79.1+-1.0 |
80.6+-0.9 |
81.6+-1.4 |
78.7+-1.1 |
||
DEVELOPMENTAL TOXITITY |
|||||
Number corpora lutea/dam |
14.35+-0.62 |
13.88+-0.49 |
13.96+-0.51 |
13.93+-0.54 |
|
Implantation sites/litter |
14.85+-0.53 |
14.68+-0.41 |
14.58+-0.66 |
14.00+-0.61 |
|
% preimplantation loss |
3.39+-1.11 |
2.53+-1.52 |
5.44+-3.33 |
5.38+-2.35 |
|
Resorptions/litter |
0.46+-0.14 |
0.28+-0.09 |
0.50+-0.18 |
0.32+-0.12 |
|
% Resorptions/litter |
3.76+-1.21 |
1.85+-0.61 |
3.25+-1.15 |
5.29+-3.58 |
|
No. litter with resorptions (%) |
9(34.6) |
7(28.0) |
8(30.8) |
7(25.0) |
|
Late fetal deaths/litter |
0.00+-0.00 |
0.00+-0.00 |
0.04+-0.04 |
0.00+-0.00 |
|
(%) Late fetal deaths/litter |
0.00+-0.00 |
0.00+-0.00 |
3.85+-385 |
0.00+-0.00 |
|
No. litter with fetal death (%) |
0(0.0) |
0(0.0) |
1(3.8) |
0(0.0) |
|
No. nonlive implants/litter |
0.46+-0.14 |
0.28+-0.09 |
0.54+-0.18 |
0.32+-0.12 |
|
(%) nonlive implants/litter |
3.76+-1.21 |
1.85+-0.61 |
7.10+-3.89 |
5.29+-3.58 |
|
No. litter with nonlive implants (%) |
9(34.6) |
7(28.0) |
9(34.6) |
7(25.0) |
|
No. liiters with live fetuses |
26 |
25 |
25 |
27 |
|
Live fetuses per litter |
14.38+-0.59 |
14.40+-0.40 |
14.60+-0.39 |
14.19+-0.44 |
|
Males per litter |
7.23+-0.47 |
6.54+-0.51 |
7.28+-0.34 |
6.96+-0.50 |
|
Females per litter |
7.15+-0.45 |
7.76+-0.52 |
7.32+-0.36 |
7.22+-0.47 |
|
% Males per litter |
50.17+-2.34 |
46.06+-3.28 |
49.96+-2.07 |
48.80+-2.96 |
|
Average fetal bw |
3.439+-0.056 |
3.583+-0.053 |
3.625+-0.054 |
3.573+-0.046 |
|
Average male fetal bw |
3.515+-0.054 |
3.689+-0.057 |
3.713+-0.057 |
3.628+-0.049 |
|
Average female fetal bw |
3.366+-0.061 |
3.502+-0.047 |
3.540+-0.050 |
3.512+-0.045 |
|
Gross malformations/litter |
0.00+-0.00 |
0.00+-0.00 |
0.04+-0.04 |
0.07+-0.05 |
|
Visceral malformations/litter |
0.15+-0.07 |
0.12+-0.07 |
0.12+-0.09 |
0.07+-0.05 |
|
Skeletal malformations/litter |
0.12+-0.12 |
0.08+-0.08 |
0.16+-0.12 |
0.07+-0.07 |
|
Fetuses malformed/litter |
0.27+-0.13 |
0.20+-0.13 |
0.32+-0.15 |
0.22+-0.12 |
|
(%) litter with malformations |
19.23 |
12.00 |
20.00 |
14.81 |
|
(%) males malformed/litter |
1.19+-0.83 |
1.19+-0.82 |
1.67+-1.18 |
2.13+-1.09 |
|
(%) females malformed/litter |
1.96+-1.19 |
1.78+-1.39 |
2.62+-1.30 |
0.74+-0.74 |
|
** p-value = 0.01(Dunnett´s test)
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 3 570 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rabbit
- Quality of whole database:
- Justification for NOAEL read-across: The potential human health hazard of the two phthalates (target and source substance) originates from the structural component of esterified phthalic acid present in both molecules. Moreover, both substances share toxicokinetic processes. Details can be found in the attached read across justification.
Additional information
DMP was evaluated for developmental toxicity in timed-pregnant Sprague-Dawley (CD) rats according to OECD TG 414 [NTP, 1989]. On gestational days (GD) 6 through 15, dietary concentrations of 0.0, 0.25, 1.0 and 5.0 % (nominal concentration) of DMP were administered, resulting in an estimated average food intake of 0.0, 0.2, 0.8 and 3.6 g/kg bw/day. High-dose DMP resulted in transient reductions in food and water consumption and body weight during early treatment. When treatment ended, food and water consumption rose above controls. Animals fed 5.0% DMP also exhibited reduced body weight gain during the treatment period and increased relative liver weight. Neither 0.25% nor 1.0% DMP treatment had significant maternal effects, with the exception of reduced water consumption during early treatment and increased food and water consumption following treatment (1.0% only). These results suggest that the apparent toxic effects of high-dose DMP on body weight may reflect the unpalatability of DMP in feed. DMP treatment during organogenesis produced no apparent effects on measured endpoints of embryo/fetal viability, growth or development. The implantation sites and percentages of resorptions, dead fetuses, nonlife implants or malformed fetuses per litter were similar among dose groups. DMP treatment had no effect on the number of live fetuses per litter or average fetal body weight per litter. There was no effect of treatment on the incidence of external, skeletal or visceral malformations. In summary, in this study in CD rats, maternal toxicity was observed at a dietary treatment level of 5.0% DMP. No developmental endpoints were affected under the conditions of this study. The NOAEL for maternal toxicity was 1.0% (840 mg/kg bw/day) DMP. The NOAEL for developmental toxicity was 5.0% (3570 mg/kg bw/day) DMP.
In a second study (Hansen, 1989), in which DMP was dermally applied to 20 -25 rats per group following a protocol similar to OECD 414, no teratogenic effects were observed. The number of copora lutea, uterus weight, implantations sites, number of live and dead foetuses, and sex distribution was unchanged. There were also no differences in the number of malformations and sceletal or visceral abnormalities. The only treatment related effect was reduced body weight gain in high dose maternal animals. Consequently, the NOAEL for developmental toxicity equals the highest dose tested of 2400mg/kg, while the NOAEL for maternal toxicity was 1200mg/kg.
The study by Gray (2000) was designed to evaluate antiandrogenic and androgenic effects. 750mg/kg DMP was administered daily via gavage to rats from gestational day 14 to postnatal day 3. Development of the male reproductive system was not different in treated and control animals, including the external genitalia (cleft phallus, hypospadias, vaginal pouch, and anogenital distance), areola and nipple retention, development of the ventral prostate, seminal vesicles, epididymides, levator ani plus bulbocavernosus muscles, and gubernactila. DMP thus has no antiandrogenic or androgenic effect in rats.
In three screening studies, the number of live and dead offspring in mice and rats were unaltered by DMP treatement. Plasterer (1985) treated 10 mice per group from gestation days 7 -14 with 3500mg/kg via gavage. Additionally, pub weight on PND 3 was evaluated and was unaffected by treatment in this study. Hardin (1987) also treated mice via gavage with up to 5000mg/kg from gestation days 6 -13. Birthweight, pub survival, litter size, and maternal weight gain were comparable to control animals. The study by Peters (1973) has lower validity due to small group sizes of only 5 rats per group, of which one animal each in the mid and low dose was not pregnant, further reducing the number of animals to be evaluated. DMP was intraperitoneally injected on days 3, 6, and 9 of pregnancy at doses of 600, 1200, and 2400mg/kg. For unknown reasons, 3 mid dose animals died, while all low and high dose animals survived. Litter sizes were slightly smaller in each treatment group compared with untreated controls, but these differences did not achieve statistical significance (app. 9 pubs per litter in treated animals vs. app. 10 in control animals).
Singh et al. [1972] is the only group that reported embryo lethality and malformations in the rat after exposure to 400, 800, or 1340mg/kg DMP (i.p., on days 5, 10, 15 of pregnancy). This study did not follow any guideline and also used only 5 animals per group. Reporting was very limited. There were no details on the observed malformations, it is unclear which pubs were affected and in which litters the effects occured. All data are provided in summary tables only. Maternal toxicity has not been assessed (incl. lacking data on mortality and final number of litters used for the assessment). Due to these deficiencies and because the effects could not be reproduced in all other studies including two studies according to or similar to OECD guideline 414 (the NTP study also adhered GLP), the results are considered unreliable and are disregarded for the assessment.
There is no developmental study for DMP available in a second species. Nevertheless, there is one study for the read-across substance DEP available. The developmental toxicity of DEP was investigated in a second species using a method equivalent to OECD TG 414 (43). Groups of pregnant female New Zealand White rabbits were treated dermally with the test substance during day 6 -18 of gestation to assess the fetotoxic and/or teratogenic effects in rabbits during organogenesis. During exposure, 12 animals per group were treated daily with 2 mL/bw of suspensions of 5%, 15%, 50% test substance corresponding to approximately 100, 300 and 1000 mg/kg bw/day. Dams were sacrificed on day 29 and fetuses were delivered by Caesarian section and examined. The number of fetuses at birth, status, distribution in the uterus and the placental indicators were recorded. The 15% and 50% dose groups showed slight skin irritations, mainly erythema. No signs of a fetal toxicity by the test substance were found in the evaluated parameters. No signs of maternal systemic toxicity and no adverse effects on the fetal development were observed in the highest dose group. The observed malformations were within the spontaneous malformation rate of the strain.
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GHS classification: no classification required
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