Registration Dossier

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:
12 Feb 2019 - 16 Oct 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Justification for study design:
Basis for dose level selection:
In a preliminary screening study with wistar rats no adverse effects were observed up to the limit dose (1000 mg/kg bw/d).

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 2-methylbutyl acetate and pentyl acetate
EC Number:
908-918-1
Molecular formula:
Unspecified
IUPAC Name:
Reaction mass of 2-methylbutyl acetate and pentyl acetate
Test material form:
liquid
Details on test material:
Name of test substance: Reaction mass of 2-methylbutyl acetate and pentyl acetate
Test substance No.: 14/0717-2
Batch identification: Tk25320180913
Purity: 96.7 area % (GC, DB-1)
99.8 area-% (GC, CP-Wax)
Storage stability: Expiry date: 12 Mar 2020
Physical state/Appearance: Liquid/ colorless, clear
Storage conditions: Room temperature
Specific details on test material used for the study:
The various analyses:
• Demonstrated the stability of the test substance preparations over a period of 7 days at room temperature (including stability over 3 days in the refrigerator).
• Confirmed the homogeneous distribution of the test substance in 0.5 % CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL)
• Confirmed the overall accuracy of the prepared test substance concentrations

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female Wistar rats, strain Crl:WI(Han), supplied by Charles River Laboratories, Research Models and Services, Germany GmbH which were free from any clinical signs of disease, were used for the investigations. The females were nulliparous and non-pregnant at the beginning of the study. The receipt of males (about 77 - 90 days old) and females (about 70 - 76 days old) at different age warrants that no sibling males and females will be paired during the study. These animals were used as F0 generation parental animals. All other animals used in this study (F1 generation pups) were derived from the supplier-provided animals.

HOUSING AND DIET
During the pretreatment period of the study, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Hohenpeißenberg, Germany.
During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
• Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
For enrichment wooden gnawing blocks (Typ Lignocel® block large, new name: SAFE® block large J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany) were added. In addition in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX B.V., Elst, Netherlands) were added. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured. The animals were housed in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20-24°C and a range of relative humidity of 45-65%. The air change rate was 15 times per hour. There were no or only minimal deviations from these limits. The day/night cycle was 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h. The animal room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection) before use. Walls and floor were cleaned once a week with water containing an appropriate disinfectant. The food used was ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Garanovit AG, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day before necropsy). Drinking water was supplied from water bottles (ad libitum). Dust-free wooden bedding was used in this study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
Test substance preparations in 0.5% CMC suspension in deionized water (with 10 mg/ 100 mL Cremophor EL)
Details on exposure:
The test substance suspensions in 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor EL were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration suspensions the specific amount of test substance was weighed in a calibrated Erlenmeyer flask, topped up with cooled 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor EL and intensely mixed with the magnetic stirrer. Afterwards the test substance preparations were kept cool in the refrigerator. For the daily administration, the test substance preparations were filled in smaller portions which were kept homogeneous with a magnetic stirrer at room temperature. During the administration the Erlenmeyer flask was closed with a plastic plug with a small hole and the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
According to preliminary test substance preparation analyses, the test substance is not homogenously soluble in water, therefore 0.5% CMC suspension in deionized water with Cremophor EL (10 mg/100mL) was chosen as solubilising agent
Details on mating procedure:
The male and female rats were about 10 - 12 weeks old, when they arrived from the breeder. During an acclimatization period of about 3 weeks, estrous cycle determination prior to treatment was performed in a pool of up to 50 non-randomized female animals. Animals with no regular estrous cycle and/or animals with the lowest and highest body weights were eliminated from the study and used for other purposes. The 40 male and 40 female animals included in the study were about 13 - 15 weeks old at the beginning of treatment and their weight variation did not exceed 20 percent of the mean weight of each sex. In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 6.30 - 9.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1"
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. Analytical verifications of the stability of the test substance in 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor EL for a period of 7 days at room temperature and refrigerator (+4°C) had been initiated prior to the start of the study. At the beginning (during premating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time points additionally one sample from the mid concentration was taken for concentration control analysis. All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Laboratory Reproduction Toxicology. The samples collected at the beginning of the administration period and during lactation were analyzed in the Analytical Laboratory. The samples of the gestation were not analyzed because no relevant imprecision occurs during the analysis of the samples from the beginning and lactation of the study. The samples within this study were labeled with serial numbers. The same number was not used for several samplings. Reserve samples were labeled with 1R, 2R, etc. The reserve samples were stored at the Laboratory Reproduction Toxicology frozen (at -20 °C) Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of/damage to original samples after agreement by the Study Director.

RESULTS
The stability of test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) was demonstrated for a period of 7 days at room temperature (including stability over 3 days in the refrigerator).
The homogeneous distribution of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) was demonstrated.
Measured values for Reaction mass of 2-methylbutyl acetate and pentyl acetate were in the expected range of the target concentrations (90 – 110 %) demonstrating the correctness of the preparations. For the low dose (100 mg/kg bw/d), mean values of sample No. 30 - 32 and their overall mean value of around 89 % at one time point was marginally below the range of 90 - 110 %. However, the test substance preparation was homogeneously distributed for this dose level and all other values of the low dose were clearly within the tolerance range. Therefore, the overall measured values of the low dose were assessed to be in an acceptable range of the nominal concentrations in this study.
Duration of treatment / exposure:
After the acclimatization period, the test substance was administered to the parental animals orally by gavage, once daily at approximately the same time in the morning. Females in labor were not treated. The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated with the vehicle (0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor EL), in the same way. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.

The duration of treatment covered a 30 days in-life period in males (including premating, mating [mating pairs were from the same test group] and postmating period) and a 2-weeks premating and mating period, the entire gestation and approximately 3 weeks of lactation period in females. Parental females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or 13.

The male and female animals were sacrificed 30 and 56 days, respectively, after the beginning of the administration, and examined.
Frequency of treatment:
Once daily
Details on study schedule:
Standardization of litters (F1 generation pups)
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Surplus pups were sacrificed. Standardization of litters was not performed in litters with ≤ 8 pups.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Mortality
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.

Clinical observations
A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and between 2 and 5 hours after the administration. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

Water consumption
Generally, water consumption was determined once a week (each time for a period of 3 days) for the male and female parental animals, with the following exceptions:
• Water consumption was not determined after the 2nd premating week (male parental animals)
• Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7, 9-10 and 12-13.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

Food consumption
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals).
• Food consumption of the females with evidence of sperm was determined on GD 0 - 7, 7 - 14 and 14 - 20.
• Food consumption of the females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

Body weight data
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results.
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 1, 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly. These body weight data were solely used for the calculations of the dose volume; therefore, these values are not reported in the summary.

Detailed clinical observations
Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of
severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed:
1. Abnormal behavior in “handling”
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos (Protruding eyeball)
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

Functional observation battery
A functional observational battery (FOB) was performed in first surviving 5 male and selected surviving 5 female animals with litter per group at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait
6. Other findings

Open field observations
The animals were transferred to a standard arena (50 × 50 × 25 cm) and observed. The following parameters were examined:
1. Behavior on removal from cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/stereotypy
14. Gait
15. Activity/arousal level
16. Feces (appearance/ consistency) within 2 minutes
17. Urine (amount/color) within 2 minutes
18. Rearing within 2 minutes
19. Other findings

Sensory motor tests/Reflexes
The animals were removed from the open field and subjected to following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Other findings
12. Grip strength of forelimbs
13. Grip strength of hindlimbs
14. Landing foot-splay test

Motor activity measurement
The measurement of motor activity (MA) was measured at the end of the administration period in first surviving 5 male and selected surviving 5 female animals with litter per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.
Oestrous cyclicity (parental animals):
For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization. For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Litter observations:
Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.

Pup body weight data
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters. In the summary tables pup body weights and pup body weight gains are listed for males, females and males + females. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Anogenital distance
Anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.

Nipple/areola anlagen
All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen was counted.
Postmortem examinations (parental animals):
Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Epididymides
3. Ovaries
4. Prostate (ventral and dorsolateral part together, fixed)
5. Seminal vesicles with coagulating glands (fixed)
6. Testes
7. Thyroid glands (with parathyroid glands) (fixed)
8. Uterus with cervix

The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):
1. Adrenal glands (fixed)
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus (fixed)

All paired organs were weighed together (left and right).

Organ/tissue fixation
The following organs or tissues of all parental animals were fixed in in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Esophagus
12. Epididymides (modified Davidson’s solution)
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Testes (modified Davidson’s solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus
48. Vagina

The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI E (1964)). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.
Postmortem examinations (offspring):
Pup necropsy observations
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone
concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.
Statistics:
Water consumption (parental animals), food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance, anogenital index:
Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means

Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups:
Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions

Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development:
Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians

Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival index:
Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians

% live male day x, %live female day x:
Comparison of the dose group with the control group was performed using the WILCOXON test (twosided) for the hypothesis of equal medians

Number of cycles and Cycle Length, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity:
Non-parametric oneway analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the equal medians
Reproductive indices:
Male reproduction data

Male mating index (%) = number of males with confirmed mating / number of males placed with females x 100
Male fertility index (%) = number of males proving their fertility / number of males placed with females x 100

Female reproduction and delivery data

Female mating index (%) = number of females mated / number of females placed with males x 100
Female fertility index (%) = number of females pregnant / number of females mated x 100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x 100
Live birth index (%) = number of liveborn pups at birth / total number of pups born x 100
Postimplantation loss (%) =number of implantations – number of pups delivered / number of implantations x 100


Sex ratio = number of live male or female pups on day 0 and 13 / number of live male and female pups on day 0 and 13 x 100
Anogenital index = anogenital distance [mm] / cubic root of pup weight [g]
Offspring viability indices:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 were determined. Pups which died accidentally or had to be sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated according to the following formulas:

Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth
* before standardization of litters (i.e. before culling)

Survival index (%) = number of live pups on day 13 after birth / number of live pups on day 4* after birth x 100
* after standardization of litters (i.e. after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All males and females of the high-dose (1000 mg/kg bw/d) and two out of ten males of the middose (300 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period.
No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the test groups (1 - 3; 100, 300 and 1000 mg/kg bw/d) during the study.
One control female showed vaginal discharge (color: red) on GD 13. This observation was not considered to be associated with the test compound.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights of all male and all female parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study.
Mean body weight change of the high-dose parental males was statistically significantly below the concurrent control values during in-life days 7 - 13, 21 - 28 and 0 - 28. Since the body weight change was only marginally decreased, it was assessed as not treatment-related. The slight, statistically significant decrease of the mid-dose parental males during in-life days 0 - 7 was assessed as not treatment-related since it was not related to dose.
Mean body weight change of all test substance-treated females and of the mid- and low-dose males was comparable to the concurrent control values during the entire study
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of all male and all female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
At the end of the administration period in females of test group 2 (300 mg/kg bw/d) platelet counts were significantly increased, but the alteration was not dose-dependent. Therefore, it was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
At the end of the administration period in males of test groups 1 and 2 (100 and 300 mg/kg bw/d) glucose levels were significantly decreased, but the change was not dose-dependent. Therefore, it was regarded as incidental and not treatment-related.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Home cage observations
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

Open field observations
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.

Sensorimotor tests/reflexes
There were no test substance-related findings in male and female animals of all test groups. One out of five examined female animals of dose group 1 showed vocalizations always when touched. Since this was not related to dose, it was assessed as spontaneous.

Quantitative Parameters
No test substance-related impaired parameters were observed in male and female animals of all test groups. The grip strength of forelimbs in females of dose group 3 was statistically significantly above the concurrent control values (8.0 versus [vs.] 5.1 in control). However, the mean value of the high-dose females was well within the range of the historical control data (HCD, GS F, mean value, range: 4.2 – 12.1) and was, therefore, assessed as incidental, not treatment-related and not adverse.

Motor activity measurement
No treatment-related, adverse changes on motor activity data (summation of all intervals) was observed in the male and female animals of all test substance-treated groups in comparison to the concurrent control values. The mean number of beam interrupts of the high-dose parental females was statistically significantly below the concurrent control values during interval 5. The numbers of beam interrupts of the intervals 1, 2 and 8 of the high-dose females were, however, above the control values. The decreased mean value of the high-dose females during one single interval was most likely an outlier since the summary value of intervals 1- 12 of the high-females was comparable to the control value. Therefore, the isolated decrease in interval 5 was assessed as incidental and not-treatment related. The statistically significantly increased number of beam interrupts in the low-dose females during interval 11 was considered to be spontaneous in nature and not treatment related since there was no relation to dose.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. This includes the 2 macroscopically detected foci in the glandular stomach of test group 2 females which correlated histopathologically to erosion/ulcer.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
In parental males (test groups 1, 2 and 3; 100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration was similar: 3.9 / 3.9 / 3.8 and 3.9 days in test groups 0 - 3, respectively.
In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (0 - 3). Fertility was proven for all F0 parental males within the scheduled mating interval for F1 litter. Thus, the male fertility index was 100% in all test groups.
The female mating index calculated after the mating period for F1 litter was 100% in all test groups (0 - 3).
The mean duration until sperm was detected (GD 0) varied between 1.5 and 3.1 days without any relation to dosing.
All female rats delivered pups or had implants in utero: The fertility index was 100% in all test groups.
The mean duration of gestation values varied between 21.9 (control), 22.6** ([**p<=0.01] test group 1), 22.0 (test group 2) and 22.2 (test group 3). The statistically significantly increased number of gestation days in the low-dose females showed no relation to dose and was considered to be spontaneous in nature and not treatment related.
The gestation index was 100% in the control and test groups 2 - 3 and 90% in test group 1.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.1 / 11.0 / 11.4 and 12.3 implants/dam in test groups 0 - 3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (11.6 / 11.6 / 11.1 and 11.3 pups/dam in test groups 0 - 3, respectively)
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.1% / 98.1% / 98.2% and 100% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.
Thus, the test substance Reaction mass of 2-methylbutyl acetate and pentyl acetate did not adversely affect reproduction and delivery of the F0 generation parental females.

Details on results (P0)

Fertility
All female animals were pregnant.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
One high-dose male pup (No. 8 of dam No. 132) had a thread-like tail during PND 0 - 4. This observation was not considered to be associated with the test compound since only one single pup was affected.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
The viability index indicating pup survival during lactation (PND 0 - 4) varied between 100%/ 98.2% / 99.2% and 100% in test groups 0 - 3, respectively.
The pups surviving index indicating pup survival during lactation (PND 4 - 13) was 100% in all test groups. Thus, the test substance did not influence pup survival in any of the treated groups (100, 300 and 1000 mg/kg bw/d).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weights and body weight change of all male and female pups in all test
substance-treated groups were comparable to the concurrent control values throughout the
entire study.
Three male runts were seen in the control, one male runt was seen in test group 1 and one
female runt was seen in test group 2.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few pups showed spontaneous findings at gross necropsy, discolored testis, thread-like tail, dilated renal pelvis, dilated ureter, hydroureter and hydronephrosis.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Thyroid hormones
In female pups at PND 13 (test groups 11, 12 and 13; 100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed. On PND 13 in male pups of test group 12 (300 mg/kg bw/d) T4 values were significantly increased, but the change was not dose-dependent. TSH values among these individuals were not altered as well as T4 and TSH values in test groups 11 and 13 (100 and 1000 mg/kg bw/d). Therefore, this T4 change in PND 13 male pups of test group 12 was regarded as incidental and not treatment-related.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of Reaction mass of 2-methylbutyl acetate and pentyl acetate by gavage to male and female Wistar rats resulted in no signs of systemic toxicity up to limit dose of 1000 mg/kg bw/d. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was the highest tested dose of 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 1000 mg/kg bw/d.