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REACH

Dimethyl phthalate

EC number: 205-011-6 | CAS number: 131-11-3

Life Cycle description
Uses advised against

Toxicological information

Toxicity to reproduction

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Administrative data

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

Cross-referenceopen all close all

Cross-reference 1

Reason / purpose for cross-reference:: reference to same study

Reference

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:

GLP compliance:

yes

Limit test:

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:

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)
Group	Inclusion level (ppm)	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)
Group	Inclusion level (ppm)	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).

Cross-reference 2

Reason / purpose for cross-reference:: reference to same study

Reference

Endpoint:

developmental toxicity

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

supporting 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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

GLP compliance:

yes

Limit test:

Specific details on test material used for the study:

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

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.

Route of administration:

oral: feed

Vehicle:

other: powdered rodent diet (4RF21 Mucedola s.r.l, Settimo Milanese (MI), Italy)

Details on exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

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:

Maternal 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

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.

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.

GROSS PATHOLOGY: Yes (see table 1)

HISTOPATHOLOGY: Yes (see table 1)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Blood sampling:

- Yes, for clinical chemistry, haematology, coagulation parmameters and thyroid hormones of parental animals and thyroid hormones of pups

Fetal examinations:

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

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:

Indices:

Presentation of data

Group mean values were calculated for all parameters. The following reproductive indices were calculated:

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

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

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.

Historical control data:

see attachment

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):

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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):

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Other effects:

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.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation periods were similar in treated groups and controls. All pregnant dams gave birth on Day 22 post coitum (mean value).

Changes in number of pregnant:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 1 009 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

other: No adverse treatment-related effects observed up to the highest tested dose.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed.

Anogenital distance of all rodent fetuses:

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.

Changes in postnatal survival:

effects 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.

External malformations:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 1 009 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse treatment-related effects on pups up to the highest tested dose.

Abnormalities:

no effects observed

Developmental effects observed:

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)
Group	Inclusion level (ppm)	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)
Group	Inclusion level (ppm)	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