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Administrative data

Description of key information

DMP:
Mouse, 1-year, dermal, similar to OECD 453 (GLP): No histopathologic changes: NOAEL 2700 mg/kg (NTP 1993)


 


Rat, oral, OECD 422 (GLP): no adverse effects observed, NOAEL 1007 mg/kg bw (males) and 1595 mg/kg bw (females) (BASF 2023)


RA to DEP (CAS 84-66-2):
Rat, 90days, oral (feed), similar to OECD 408 (no GLP): red. b.w. (gain) and food consumption, increased liver weight: NOAEL = 750mg/kg (Brown 1977)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
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: 10 - 11 weeks old
- Weight at study initiation: 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
Details on route of administration:
The oral route was selected as it is a possible route of exposure of the test item in man and has been specifically requested by the Regulatory Authorities.
Vehicle:
other: powdered rodent diet (4RF21 Mucedola s.r.l, Settimo Milanese (MI), Italy)
Details on oral 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
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
Observations and examinations performed and frequency:
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:

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.

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.

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.

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.

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.



Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 1)

HISTOPATHOLOGY: Yes (see table 1)
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.
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 treatment-related changes were observed in absolute and relative organ weights of treated animals, when compared to the controls.
There were no treatment-related changes in terminal body weight and organ weights when compared to the controls. The statistically significantly lower in absolute and relative mean prostate weights in treated males (-17% for Group 2, -19 % for Group 3 and -16 % for Group 4 relative to body weight) did not show a dose- dependency and the values were in the historical control range of the rat strain used. Additionally, there were no of corresponding microscopic observations. Thus, these differences were considered not to be related to treatment. Any organ weight changes other than that listed above were within the range of occasionally observed and expected spontaneous changes in rats of the same age and considered unrelated to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed at post mortem examination in treated animals, when compared to the controls.
Neuropathological findings:
no effects observed
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).
Other effects:
effects observed, non-treatment-related
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.

Thyroid hormone determination (T4 and TSH):
- Adult males: No treatment-related changes were recorded in parental males.
- Pups: No relevant changes were recorded in pups of both sexes at Day 14 post partum.

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.


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.

Litter data at birth, on Day 1, on Day 4 (before culling) on Day 7 and on Day 13 post partum
of females and sex ratio of pups:
- Litter data: 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.
- Litter data - Day 0 to Day 4 post partum - before culling and on Day 7 post partum: At birth and on Day 1 post partum, no treatment-related differences were observed in total litter size, live litter size, mean pup loss, litter weight and mean pup weights among the treated dams and the controls. 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.
- Litter data - Day 13 post partum - after culling: Litter weight and mean pups weight (for both sexes combined) of treated groups were similar to controls. No differences in pup loss were noted between control and treated groups.
- Sex ratio: 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.

Clinical signs of pups.
- No treatment-related clinical signs were observed in pups during the 14-day observation period.

Anogenital distance:
- No differences in the anogenital distance (normalised value) performed on Day 1 post partum, were noted between control and treated pups.

Pups thyroid weight on Day 14 post partum:
- No significant differences were observed in the weight of the thyroid in treated pups, when compared to controls.

Necropsy findings in pups and nipple check:
- 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.
- No nipples were observed in male pups on the day of necropsy.

Details on results:
DISCUSSION:

The toxicity effects of the test substance, 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 this study, 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). The vehicle was powdered rodent diet.

Males were treated for 2 consecutive weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 34 days. Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a period of at least 51 days.
No mortality occurred throughout the study and no treatment-related clinical signs were noted during the study.
No signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reactivity to stimuli) 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 (including coagulation and blood clotting time) 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 (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 1007mg/kg/day for males and 1595mg/kg/day for females).
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

Table. 2: Summary details of the pregnant status


 






























































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


 






































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


 










































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



 


 


 

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:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable study that meets basic scientific principles. However, study was performed in 1977 and is a non-GLP and non-guideline study but well documented publication.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
Groups of 15 male and 15 female rals were given diets containing 0 (control), 0.2, 1.0 or 50 % diethyl phthalate (DEP) for 16 wk.
GLP compliance:
no
Remarks:
times before GLP
Specific details on test material used for the study:
- Name of test material (as cited in study report): DEP, diethyl phthalate
- Analytical purity: min. 99%
- Impurities (identity and concentrations): phthalaic acid max. 0.01 %, ash max. 0.01 %; water max 0.1 %
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: CD rat (Sprague-Dawley derived); supplier: Charles river U.K., Kent
- Age at study initiation: no details reported
- Weight at study initiation: no details reported
- Fasting period before study: no details reported
- Housing: animal room
- Diet (e.g. ad libitum): Spratts Laboratory Diet No. 1
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no details reported


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 50-60
- Air changes (per hr): no details reported
- Photoperiod (hrs dark / hrs light): no details reported

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- test substance was apllied with diet: 0, 0.2, 1.0 and 5.0 % in diet
Duration of treatment / exposure:
16 weeks
Frequency of treatment:
daily
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
male, 0.2 % nominal in diet
Dose / conc.:
770 mg/kg bw/day (actual dose received)
Remarks:
male, 1.0 % nominal in diet
Dose / conc.:
3 160 mg/kg bw/day (actual dose received)
Remarks:
male, 5.0 % nominal in diet
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
female, 0.2 % nominal in diet
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
female, 1.0 % nominal in diet
Dose / conc.:
3 710 mg/kg bw/day (actual dose received)
Remarks:
female, 5.0 % nominal in diet
No. of animals per sex per dose:
- 15 in the main study
- 5 in a parallel study where similar diet preparations were fed
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: days 1, 27, 56, 112

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: days 1, 27, 56, 112

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study
- Anaesthetic used for blood collection: Yes (barbiturate)
- Animals fasted: Yes, dprived of food one night prior to sacrifice
- How many animals: all naimals, 15 per sex and dose
- Parameters checked in table [No. 2] were examined.

CLINICAL CHEMISTRY: Yes
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 2,3,
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.2] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 1)
HISTOPATHOLOGY: Yes (see table 1)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- no substance-realated changes in behaviour patterns nor other clinical signs of toxicity

BODY WEIGHT AND WEIGHT GAIN (see table 3 for details)
- Final body weight was reduced in both sexes given 5% DEP in the diet (females: - 20.4%, males: - 23%)
- A reduction of body-weight was seen also in female rats fed 1% DEP (-9%)
- A transient but statistically significant effect was apparent in male rats given the 1 % dietary level from day 6 to 36.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- matching the reduced body weights, food intake was lower in high dose males and females. The difference reached significance at the beginning of the study and when comparing the mean food consumption. Since food consumption increased over time, the reduction is likely due to the bad taste of the diet containing DEP.
- Mean food consumption was also significantly reduced for mid dose females, but values obtained at intermediate time points did not reach significance. This matches the less severe effect on final body weight. The difference was also most pronounced at the beginning, with values comparable to controls at the end of the study.

WATER CONSUMPTION
- no statistically significant substance-related effects observed

HAEMATOLOGY
- Erythrocyte count was significantly higher in male rats given 5 % dietary DEP for 6 week than in the controls
- accompanied by a hemoglobin level higher than but not significantly different from the control value
- no siginficant alteration in blood enzyme activity

URINALYSIS
- no significant increasc in urinary cell excretion in males or females in any dose group; male rats given 5% DEP exereted significant fewer cells in the 13th week;
- no statistically significant differences
- During an 2h urine dilution test at week 6, male rats receiving 5% DEP in diet excreted a significantly larger volume than controls, female rats excreted a significantly smaller volume

ORGAN WEIGHTS (see table 4 for details on relative organ weights and teminal body weights; % weight increase/decrease refers to control and terminal organ weight, if not indicated differently)
- rel. brain weight was increased in M and F in the 5.0 % dose group at weeks 2,6, 16 (+28% in M, +20 %in F)
- rel. liver weight was increased in M and F in the 5.0 % dose group (+33% in M, +31%in F). In females, liver weights were increased in all dose groups
- rel. kidney weight was increased in M and F in the 5.0 % dose group (+18% in M, +11%in F)
- Relative stomach and/or small intestinal weights were increased in mid and high dose males and all treated females. These differences were due to unusual low control values compared to historical controls rather than a substance specific effect.
- rel. full caecum weight was increased in M and F in the 5.0 % dose group at all at week 16, and empty caecum was increased inly in F in the high dose group
- rel. adrenal weight was increased only in M in the 5.0 % dose group at week 16 (+16% in M)
- rel. gonad (testis) weight was increased in M in the 5.0 % dose group at weeks 2,6, 16 (+29% in M); there were no effects on F gonads
- rel. pituitary and thyroid gland weights were increased only in M in the 5.0 % dose group (+19% and +17%, respectively)

GROSS PATHOLOGY
- only one unilateral small testis without histopathological substance-related findings
- no statistically significant substance-related effects observed

HISTOPATHOLOGY: NON-NEOPLASTIC
- no statistically significant substance-related effects observed

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
- no statistically significant substance-related effects observed
Dose descriptor:
NOAEL
Effect level:
770 mg/kg bw/day (actual dose received)
Sex:
male
Remarks on result:
other: 1.0% in diet
Dose descriptor:
LOAEL
Effect level:
3 160 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
body weight and weight gain
Remarks on result:
other: 5.0% in diet
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Sex:
female
Remarks on result:
other: 1.0 % in diet
Dose descriptor:
LOAEL
Effect level:
3 710 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: 5.0 % in diet
Critical effects observed:
no

Table 3: results from the 90 day DEP feeding study in Sprague-Dawley rats

Dietary levels %

Male

Female

0

0.2

1.0

5.0

0

0.2

1.0

5.0

Body weight (g) at day

0

125

125

124

124

109

109

109

109

27

356

352

334*

271*

225

235

213*

191***

56

473

476

459

356***

283

286

266*

234***

112

599

617

575

461***

358

347

328*

285***

Food consumption (g/rat/day) at day

1

15.5

17.3

13.8*

3.2

13.7

13.9

11.4

3.7*

27

27.3

29.7

30.0

25.5

18.5

19.3

16.2

16.6

56

31.3

29.5

25.9

23.8

21.9

17.1

19.3

16.1

112

21.5

21.7

21.8

20.2

15.2

15.8

14.7

14.3

Mean food consumption (g/rat/day) at day

24.9

25.3

24.7

19.1*

18.5

17.7

16.5*

15.1*

Mean water intake (g/rat/day) up to day 112

35.2

33.4

31.7

34.4

31.9

27.1

35.5

28.1

*           P 0.05

***        P 0.001

Students T-test for BW and ranking method of Wilcoxon (1945) for food consumption

Table 4: Relative organ weights

Dietary levels %

Male

Female

0.0

1.0

5.0

0.0

1.0

5.0

Week 2 (5/5 animals)

Brain

0.97

0.99

1.23*

1.15

1.20

1.31*

Heart

0.45

0.46

0.44

0.42

0.41

0.42

Liver

3.37

3.67*

4.78***

3.63

3.56

4.81***

Spleen

0.30

0.28

0.30

0.27

0.28

0.29

Kidneys

0.93

0.99

1.04*

1.00

0.99

1.06

Stomach

0.55

0.59

0.84**

0.83

0.64

0.86

Small intestine

3.66

3.67

3.97

3.35

3.29

3.79**

Caecum empty

0.42

0.46

0.53

0.44

0.48

0.49

Caecum full

1.79

1.51

2.24

1.71

1.65

2.14

Adrenals

18.4

20.3

21.5

33.4

27.8

26.1

Gonads

1.07

1.05

1.32*

61

70

57

Pituitary

3.9

3.0

3.7

5.4

5.1

4.7

Thyroid

6.8

7.3

7.5

7.8

9.9

10.9

Terminal BW (g)

194

193

149***

157

149

134***

Week 6 (5/5 animals)

Brain

0.55

0.55

0.76**

0.80

0.84

0.97**

Heart

0.33

0.34

0.35

0.40

0.41

0.41

Liver

2.56

2.94*

3.41**

2.69

2.93

3.57***

Spleen

0.18

0.21

0.22

0.26

0.26

0.23

Kidneys

0.76

0.75

0.81

0.74

0.70

0.81

Stomach

0.39

0.40

0.52*

0.46

0.48

0.59***

Small intestine

1.89

2.10

2.57***

2.76

2.58

2.78

Caecum empty

0.27

0.30

0.34*

0.40

0.45

0.42

Caecum full

0.74

0.77

1.15*

1.01

1.34

1.50**

Adrenals

16.2

13.9

17.5

28.6

29.9

30.9

Gonads

0.86

0.94

1.23**

55

60

49

Pituitary

2.9

2.6

2.8

4.2

4.2

3.7

Thyroid

4.8

4.8

6.5**

5.5

6.4

6.6

Terminal BW (g)

398

385

274***

237

231

199**

Week 16 (15/15 animals)

Dietary levels %

Male

Female

0

0.2

1.0

5.0

0

0.2

1.0

5.0

Body weight (g) at day

Brain

0.39

0.39

0.39

0.50***

0.65

0.64

0.68

0.78***

Heart

0.27

0.29

0.27

0.31**

0.31

0.32

0.33

0.32

Liver

2.22

2.16

2.29

2.95***

2.17

2.31*

2.35**

2.84***

Spleen

0.15

0.13

0.14

0.14

0.16

0.16

0.17

0.19**

Kidneys

0.57

0.58

0.57

0.67***

0.62

0.62

0.64

0.69***

Stomach

0.29

0.29

0.32*

0.41***

0.35

0.40***

0.41***

0.51***

Small intestine

1.51

1.53

1.57

1.93***

1.99

2.23**

2.26*

2.47***

Caecum empty

0.21

0.23

0.24

0.24

0.27

0.30*

0.29

0.34***

Caecum full

0.60

0.60

0.71

0.89***

0.77

0.86

0.89

1.43***

Adrenals

9.6

9.1

9.3

11.2*

19.7

19.1

20.2

22.1

Gonads

0.66

0.64

0.66

0.85***

26

27

28

28

Pituitary

2.1

2.0

2.2

2.5***

4.7

4.5

4.7

4.4

Thyroid

3.5

3.2

3.5

4.1**

4.9

5.0

5.2

5.8

Terminal BW (g)

568

585

559

438***

330

328

304*

267***

Relative organ weights are given in [g/100 g bw], except terminal bw in [g]

*           P 0.05

**         P 0.01

***        P 0.001

Students T-test for BW and ranking method of Wilcoxon (1945) for food consumption

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable study, which meets basic scientific principles
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Specific details on test material used for the study:
Dimethyl phthalate;
colorless liquid, >=99% pure;
Supplied by Chemical Technical Industries, Orlando, Florida,
Lot No.: C122883;
Stability was monitored regularly during the study period, no degradation was observed.
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles Rliver Breeding laboratories, Kingston, NY
- Age at study initiation: 7 weeks
- Weight at study initiation:32.6
- Housing: individually
- Diet (e.g. ad libitum): NIN-07 open formula meal ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 28-74%
- Air changes (per hr): at least 12
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Details on exposure:
0.1mL of the test substance was applied to the clipped interscapular skin
Duration of treatment / exposure:
1 year (55 weeks)
Frequency of treatment:
5 times/week
Dose / conc.:
2 700 mg/kg bw/day
Remarks:
based on an average weight of 44g, volume administered: 0.1 ml per mouse
No. of animals per sex per dose:
50
Control animals:
yes
Details on study design:
Control animals were treated with acetone, 3 times per week in the first 8 weeks, twice per week thereafter due to skin irritation
Post-exposure period: none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: Clinical findings, mortality

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

DERMAL IRRITATION (if dermal study): Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for the first 13 weeks, monthly thereafter
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
All gross lesions and tissue masses
Adrenal gland, brain, esophagus, gallbladder, heart, kidney, large intestine, liver, lung, mammary gland, mandibular and mesenteric lymph nodes, nose, pancreas, parathyroid gland, pituitary gland, prostate gland, salvary gland, seminal vesicles, skin (site of application and other), small intestine, spleen, sternum, stomach, testis, thymus, thyroid gland, trachea, and urinary bladder
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
>= 2 700 mg/kg bw/day
Sex:
male
Basis for effect level:
other: no adverse effects at the highest tested dose
Critical effects observed:
no
No effect on survival or growth was reported. 
No microscopic changes in a comprehensive range of tissues were reported with the exception of skin irritation at the site of application, which was observed in treated (11/49) and vehicle control (8/50) animals.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 700 mg/kg bw/day

Additional information

Repeated dose toxicty: Oral:


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 an 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; see IUCLID chapter 7.8 for more details).
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.
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.
Based on the results of the present study, the NOAEL for general 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).


There are further studies with less reliability but similar results, indicating a low toxicity after oral exposure:


Lehman et al. (1955) fed female rats 2, 4, 8% DMP in the diet for 2 years. Slight but significant effects on growth were observed at 8.0 and 4.0%, whilst there was no effect at the 2.0% level. Blood count and mortality rates in the treated animals did not differ from the controls. Kidney damage was observed at the top-dose level (chronic nephritis). The NOAEL was 2% corresponding to about 1000mg/kg b.w.


 


Kwack (2009) treated male rats with 500 mg/kg bw day for 4 weeks. No Adverse Effects were seen in all parameters examined (Clinical Signs, Mortality, Dietary Consumption, and Body and Organ Weights, Blood and Urine Analysis, Sperm Count and Motility Analysis). Increased ALP level might indicate an increased liver metabolism.


 


Bell (1982) described slightly, but significantly decreased lipid and cholesterol content in the livers of male rats fed 2.5 mmol/100g (app. 500 mg/kg) in the diet for 21 days. In combination with a non-significant increase in sterologenesis (measured as production of cholesterol), these results might indicate increased liver metabolism.


 


Repeated dose toxicity: Dermal:


In a chronic dermal study, app. 2700mg/kg b.w. of pure DMP (0.1mL per mouse) were applied to the interscapular skin of 50 mice per group, 5 times per week for 55 weeks (NTP 1993). Acetone treated mice were used as control. There was no influence on clinical signs, body weight, or mortality. Extensive histopathology of all gross lesions and a large range of organs did not reveal any differences to control animals. There was no assessment of blood or urine parameters. Chronic DMP as well as acetone (control) treatment caused skin irritation in 11 and 8 mice, respectivly. Due to the abscence of any adverse effect, the NOAEL in this study was 2700mg/kg.


 


Further publications which also describe effects of subchronic (50 -90) day dermal exposure to DMP were disregarded because none provided data on control animals and no details on severity of observed effects or statistical evaluation was provided. Most data were also only provided in reviews which were not based on peer reviewed publications.


 


To support the results of the studies described above, a subchronic study with the read across substance DEP is also provided:


 


Repeated dose toxicity: Oral: Read across to DEP:


Groups of 15 male and 15 female Sprague-Dawley rals were given diets containing 0, 0.2, 1 .0 or 5.0 % diethyl phthalate (DEP) for 16 wk corresponding to 0, 150, 770, 3160 mg/kg bw/day for males and 0, 150, 770, 3710 mg/kg bw/day for females [Brown et al. 1977]. Animals were treated for 90 days daily and body weights, food and water consumption were recorded weekly. A full necropsy was performed including histopathology.


Consumption of a diet containing 5.0 %, DEP was associated with a reduction in food intake and body weight (-20%). Similar, but less severe effects were seen in the females given 1.0 % DEP (-9%). Food intake was mainly reduced at the beginning of the study, indicating palatability issues rather than substance related effects. No statistically significant effects on water intake or on the results of the haematological examinations, serum-enzyme levels, urinary ceIl-excretion rate, renal concentration tests or histological examination were seen in the treated animals. However, there were increases at week 16 in the relative liver weight of females at all treatment levels and of males fed the highest level. In the abscence of histopathological findings, these changes are considered adaptive to increased metabolism. High dose animals of both sexes also showed increased weights of several other organs, which correlate to the reduced final body weight.


Even though effects seen at the high dose are likely secondary to reduced food intake and consequently reduced body weight, the effects are considered adverse due to their magnitude, the involvement of several parameters, and because other reasons cannot be fully excluded. The NOAEL in this study was thus set to 1% DEP in the diet, corresponding to 770 and 750mg/kg for males and females, respectively.


 


This result corresponds well to the above described results for DMP, which also show low toxicity after repeated exposure and NOAEL values of around 1000mg/kg.

Justification for classification or non-classification

GHS classification: no classification required for repeated dose target organ toxicity.