4-(1,1-dimethylethyl)cyclohexyl acrylate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A study according OECD 422 was performed. The substance was given daily by gavage as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0, 50, 150 and 500 mg/kg mg/kg bw/day. Control animals were dosed daily with the vehicle only (corn oil).

The NOAEL for fertility/development was 500 mg/kg bw/day, the highest dose tested. The NOAEL for systemic toxicity was 150 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10 - 11 weeks (males/females)
- Housing: individually (polycarbonate cages type III supplied by TECNIPLAST)
- 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 4 (end of lactation).
- Diet: ad libitum (ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum (dringing water)
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently released with a magnetic stirrer. The test substance preparations were produced weekly and stored at room temperature.

VEHICLE
- Justification for use and choice of vehicle: In the context of toxicological studies the stability of the test substance in the vehicle corn oil has to be verified.
- Concentration in vehicle: 1.25 g/100 mL (50 mg/kg bw/day); 3.75 g/100 mL (150 mg/kg bw/day); 12.50 g/100 mL (500 mg/kg bw/day)
- Amount of vehicle: 4 mL/kg bw

Details on mating procedure:

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 test group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 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 was detected was denoted gestation day (GD) 0 and the following day "GD 1".

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

In the context of toxicological studies the stability of the test substance in the vehicle corn oil has to be verified.
Concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start of the administration period. Given that the test substance is completely miscible with corn oil, solutions were considered to be homogenous and no homogeneity analyses were carried out.

Duration of treatment / exposure:

males: 28 days
female: 57 days

Frequency of treatment:

daily

Details on study schedule:

- Dose selection rationale: Dose levels were selected by request of the sponsor.
- Route of administration: The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

MORTALITY / CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.Abnormalities and changes were documented daily for each affected animal. The littering 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 public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level , tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals:
- 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 4.
- Females without a litter and without positive evidence of sperm in the vaginal smear or waiting for necropsy were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

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 during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14 and 14-20.
- Food consumption of F0 females, which gave birth to a litter was determined for PND 1-4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION:
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning towards the end of tha administrative period.
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning towards the end of tha administrative period.
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group

URINALYSIS: Yes
- Time schedule for collection of urine: overnight towards the end of the administrative period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in the first five surviving male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period
- Dose groups that were examined: all amimals
- Battery of functions tested:
Home cage observations:
The animals were observed in their closed home cages; during this period 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: Posture, tremors, convulsions, abnormal movements, impairment of gait
Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: Behavior on removal from the cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors,
convulsions, abnormal movements/stereotypes, impairment of gait, activity/arousal level, feces excreted within 2 minutes (appearance/consistency), urine excreted within 2 minutes (amount/color, rearing within 2 minutes.
Sensory motor tests/ reflexes:
The animals were then 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), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs and hindlimbs, landing foot-splay test.

Motor activity (MA) was also measured on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group.

Sperm parameters (parental animals):

stages of spermatogenesis

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups
- stillbirths
- live births
- postnatal mortality
- presence of gross anomalies
- weight gain, physical or behavioural abnormalities

BODY WEIGHT
The pups were weighed on the day after birth (PND 1) and on PND 4.

CLINICAL OBSERVATIONS
The live pups were examined daily for clinical symptoms (including grossmorphological findings) during the clinical inspection of the dams.

STANDARDISATION OF LITTERS
Pups were scheduled sacrifice on PND 4.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
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: Anesthetized animals, epididymides, tstes.
- The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations): Adrenal glands, brain, heart, kidneys, liver,spleen, thymus.

The following organs or tissues of all parental animals were fixed in in 4% neutralbuffered formaldehyde or in modified Davidson’s solution: All gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, eyes with optic nerve, esophagus, extraorbital lacrimal glands, epididymides (modified Davidson’s solution), femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries (modified Davidson’s solution), oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate gland, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes (modified Davidson’s solution), thymus, thyroid glands, trachea, urinary bladder, uterus (uteri of all cohabited female F0 parental animals were stained according to Salewski’s method), vagina.

Postmortem examinations (offspring):

All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 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:

Statistics of clinical pathology
Means, medians and standard deviations of each test group were calculated for several parameters.
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians
Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity): Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Urine pH, volume, specific gravity, color and turbidity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 hypothesis of equal medians. Urine color and turbidity are not evaluated statistically.

Statistics of pathology
Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians

Reproductive indices:

Male reproduction data
The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs. For the males, mating and fertility indices were calculated for F1 litters.
Male mating index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
*defined by a female with vaginal sperm or with implants in utero
number of males proving their fertility*
Male fertility index (%) = (number of males proving their fertility* / number of males placed with females) x 100
* defined by a female with implants in utero

Female reproduction and delivery data
The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females. For the females, mating, fertility and gestation indices were calculated for F1 litters.
number of females mated*
Female mating index (%) = (number of females mated* / number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero
number of females pregnant*
Female fertility index (%) = (number of females pregnant* / number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero
Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x 100
* defined as the number of females with implants in utero
number of liveborn pups at birth
Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100
total number of pups born
The implantations were counted and the postimplantation loss (in %) was calculated.
Post implantation loss (%) = ((number of implantations - number of pups delivered) / number of implantations) x 100

Offspring viability indices:

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 viability/mortality
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.
Viability index (%) = (number of live pups on day 4 after birth / number of live pups on the day of birth) x 100

Sex ratio:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth.
Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Tonic-clonic convulsions were observed in 5 high dose female animals on different days during lactation period. Twitching (mostly slight, sometimes moderate) was observed during the entire administration period in 9 male and all female animals of test group 3 (500 mg/kg bw/d) from premating day 8 onwards on different study days (mostly 0-2 hours, partly 2-5 hours after treatment). The findings were assessed as treatement related and adverse.
Piloerection was observed in 2 male animals of test group 3 (500 mg/kg bw/d) from mating day 2 (study day 15) onwards on different study days. The finding was assessed as being related to treatment and adverse. A single male animal of test group 2 (150 mg/kg bw/d) showed piloerection just on mating day 2. In this case, a relation to treatment was not assumed because of the transient and short occurrence of the finding. Plough nose-first into bedding was observed shortly after treatment during the entire administration period (except gestation and lactation) on different study days in all males and 4 females of test group 3 (500 mg/kg bw/d) as well as in 6 males and 1 female of test group 2 (150 mg/kg bw/d). Salivation shortly after treatment, mostly slight, sometimes moderate, was observed in all phases of the study for all male and female animals of test group 3 (500 mg/kg bw/d) as well as for 7 male and 2 female animals of test group 2 (150 mg/kg bw/d). From the temporary, short appearance immediately after dosing it was concluded that plough nose-first into bedding and salivation were induced by a bad taste of the test substance or local affection of the upper digestive tract.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight of male animals of test group 3 (500 mg/kg bw/d) was significantly reduced on mating day 8 (-5%). Mean body weight change values in male animals of test group 3 (500 mg/kg bw/d) were significantly lower between premating days 0-7 (- 53%) and 0-13 (-41%). These changes were assessed to be related to treatment and adverse.
No significant changes were observed in female animals of test group 3 (500 mg/kg bw/d) as well as in male and female animals of test groups 1 and 2 (50 and 150 mg/kg bw/d).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the premating period food consumption was significantly decreased in males of test group 3 (500 mg/kg bw/d) between study days 0-7 and 0-13 as well as in females of the same test group between study days 0-7, 7-13 and 0-13. The changes were assessed to be related to treatment. No comparable observation were made in any other test group of the study. No impairment of food consumption was observed for female animals of any test group during the gestation and lactation periods.

Water consumption and compound intake (if drinking water study):

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among hematological parameters were observed. In male rats of test group 3 (500 mg/kg bw/d) absolute neutrophil cell counts and in females of the same test group absolute monocyte cell counts were higher compared to controls. In males the absolute neutrophil counts were within the historical control range and in females the absolute monocyte counts were marginally above the range (males absolute neutrophils 0.70-1.35 Giga/L; females absolute monocytes 0.05-0.09 Giga/L; PART III, Supplement). No other changes in the differential blood cell counts occurred. Therefore, the change in males was regarded as incidental and not treatment-related, and the marginally higher monocyte counts in females were regarded as maybe treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).
In males of test group 2 (150 mg/kg bw/d) relative neutrophil cell counts were decreased and relative lymphocyte cell counts were increased. Both parameters were not dose-dependently changed and therefore this alteration was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The only treatment related effect were increased urea levels and decreased glucose levels in high dose males. These changes might be related to the alpha 2µ nephropathy observed in all dose groups, which does not represent a human relevant hazard.

In females of test groups 2 and 3 (150 and 500 mg/kg bw/day) inorganic phosphate and cholesterol levels were increased. However, cholesterol levels in both mentioned test groups and inorganic phosphate values at least in test group 2 were within the historical control ranges (cholesterol 1.13-1.77 mmol/L; inorganic phosphate 1.14-1.55 mmol/L; PART III, Supplement). Therefore, the changes in the mentioned test groups were regarded as incidental and not treatment-related. Higher inorganic phosphate levels in females of test group 3 (500 mg/kg bw/dây) were the only altered parameter in these individuals and therefore this increase was regarded as maybe treatment-related but not adverse (ECETOC Technical Report No. 85, 2002). In males of test groups 1 and 2 (50 and 150 mg/kg bw/day) total bile acid levels were lower compared to controls, but this change was not dose-dependent. Therefore, it was regarded as incidental and not treatment-related.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among urinalysis parameters were observed.

In males of test group 3 (500 mg/kg bw/day) urine pH value was lower and in females of the same test group urine volume was higher compared to controls. Both parameter changes without any other changes in the kidneys were regarded as treatment-related but not adverse.
In males of test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/day) higher incidences of transitional epithelial cells and granulated and epithelial casts were found in the urine sediment. This observation correlates well with the histopathological finding of alpha 2µ-Globulinurie, which was regarded as a rat specific effect with no relevance to humans (Hard et al., 1993).

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Home cage observations: Twitching was observed in 2 females (Nos. 137 and 138) of test group 3 (500 mg/kg bw/day).
Open field observations: Twitching was observed in 2 females (Nos. 137 and 138) of test group 3 (500 mg/kg bw/day).
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative Parameters: No test substance-related effects were observed.
Motor activity measurement: A significant deviation concerning the overall motor activity (summation of all intervals) was observed in female animals of test group 1 (50 mg/kg bw/day) in comparison to the concurrent control group. Regarding single intervals in male animals of test group 1 (50 mg/kg bw/day) a significantly reduced activity was observed in interval 6 as well as in female animals of test group 1 (50 mg/kg bw/day) in interval 11. As no other single intervals as well as the overall motor activity showed significant deviations to the control values and a dose-response relationship was not observed, the finding was assessed to be incidental and not related to treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the kidneys of male animals. The number and pattern of eosinophilic droplets in the proximal convoluted tubules was highly discerned in the H&E stained tissues. Thus, a dose-dependent increase of eosinophilic droplets was noted. This finding was characterized not only by an increase in the number of droplets in the tubular epithelium cells but also by an increase in the number of affected tubules. With increasing dose, the form of the eosinophilic droplets changed to more angular and crystal-shaped eosinophilic aggregates. Concomitantly, tubular degeneration and regeneration in the cortex and outer stripe of the outer medulla (OSOM) and granular casts at the junction of the OSOM and the inner stripe of the outer medulla (ISOM) accompanied this process. The immunostaining with an antibody to α2µ-globulin correlated partially with the distribution pattern of eosinophilic droplets. All other 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.

Histopathological findings: neoplastic:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The male mating index was 90% in control group (0 mg/kg bw/day) and 100% in test groups 1-3 (50, 150 and 500 mg/kg bw/day). The male fertility index was 80% in control and high dose rats, 90% in the low dose and 70% in the mid dose. These values reflected the normal range of biological variation inherent in the strain of rats used for this study. The non-pregnant females as well as their male mating partners did not show alterations in the histopathologic examination that could have impaired their fertility.

The female mating index calculated after the mating period for F1 litter was 90% in control group (0 mg/kg bw/day) and 100% in test groups 1-3 (50, 150 and 500 mg/kg bw/day). The mean duration until sperm was detected varied between 1.6 and 3.1 days without relation to dose and within the normal range for this strain of rats. Gestation length was similar for all dose groups.

Key result

Dose descriptor:

NOAEL

Remarks:

(parental systemic toxicity)

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

Remarks on result:

other: tubular damage in the kidneys of male animals of all test groups as a consequence of an α2 µ nephropathy does not represent a risk for humans

Key result

Dose descriptor:

NOAEL

Remarks:

(parental repoductive toxicity- fertility)

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup mortality during lactation (PND 0-4) was 98.1% in control animals, 100% in the low and mid dose and 96.7% in the high dose. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. Two pups in the control group and 4 pups in the high dose were cannibalized. One pup in the high dose group was stillborn. A relation to treatment was excluded.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group. One male runt each was found in the control and the low dose. Two female runts occurred in the mid dose. One male and one female runt were found in the high dose.

Gross pathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups.

Key result

Dose descriptor:

NOAEL

Remarks:

(developmental toxicity)

Generation:

F1

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Species:

rat

Quality of whole database:

OECD 422, GLP (RL1)

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 422

The substance was given daily by gavage as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0, 50, 150 and 500 mg/kg mg/kg bw/day. Control animals were dosed daily with the vehicle only (corn oil). The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females up to one day prior to the day of schedule sacrifice of the animals. The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the post implantation loss in all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation (days 0-7, 7-14, 14-20) and lactation (days 1-4). In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0 and 4. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

There were no effects on mating performance, or alterations in reproductive organs in parental animals. Pup number, survival, body weight, and sex ratio was comparable between all groups. No alterations were detected during gross pathology of the pups. There were, however, signs of systemic toxicity in high dose maternal animals: reduced body weight and body weight gain and twitching and tonic-clonic convulsions. α2µ nephropathy was detected in males of all groups, but was not considered relevant for human risk assessment. Details on systemic toxicity are included in the corresponding section on repeated dose toxicity.

 

Consequently, the NOAEL for reproductive performance and fertility was set to 500 mg/kg bw/day for male and female Wistar rats. The NOAEL for developmental toxicity was also 500 mg/kg bw/day, the highest dose tested.

The NOAEL for systemic toxicity (excluding effects without human relevance) was 150 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

A study according OECD 414 was performed in time-mated female Wistar Han rats. The substance was administered from Days 6 to 20 post coitum, inclusive, by daily oral gavage at dose levels of 60, 180 or 540 mg/kg bw/day. The rats of the control group received the vehicle, corn oil, alone. The following results have been obtained:

Maternal NOAEL: 180 mg/kg bw/day (based on clinical signs, lower body weight and lower food consumption observed at 540 mg/kg bw/day).

Developmental NOAEL: at least 540 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-07-11 to 2022-08-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld
- Age at study initiation: 11-15 weeks old
- Weight at study initiation: 184 – 274 g
- Fasting period before study: no
- Housing: individually housed in Polycarbonate cages (Makrolon type MIII)
- Diet: ad libitum, pellets, SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
- Water: ad libitum, Municipal tap water
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 51-73
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 Jul 2022 To: 29 Jul 2022

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations were kept at room temperature until dosing.

VEHICLE
- Justification for use and choice of vehicle: solubility
- Concentration in vehicle: 0, 15, 45 and 135 mg/mL
- Amount of vehicle: 4 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations. No test material was detected in the Group 1 formulation. The formulations of Groups 2 and 4 were homogeneous (i.e., coefficient of variation ≤ 10%).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

Day 6 to Day 20 post-coitum

Frequency of treatment:

once daily

Duration of test:

14 days

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Group 1

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Dose / conc.:

180 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Dose / conc.:

540 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses have been selected based on a dose range finding study (see supporting study).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily 1 to 2 hours post-dose, starting on Day 6 post coitum up to and including the day prior to necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Days 2, 6, 15 and 21 post-coitum
BODY WEIGHT: Yes
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION AND COMPOUND INTAKE:
Water consumption was monitored by visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: ovaries and uterus, thyroid

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Serum: Yes
- Volume collected: 1 mL
- T3, T4 and TSH analysis in serum

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes

Statistics:

Parametric/Non-Parametric:
Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons was conducted using Dunnett’s or Dunn’s test, respectively.

Non-Parametric:
The groups will be compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons was conducted using Dunn’s test.

Indices:

See "Any other information on materials and methods incl. tables"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test material-related clinical signs were noted at 60 mg/kg/day.
At 540 mg/kg/day, tremors (slight) were observed in 5/22 females on one or multiple days between Days 14-16 post-coitum and generalized twitches (slight to moderate) were noted in 13/22 females between Days 16-20 post-coitum. Moreover, erected fur was observed in 10/22 females on various days mainly during the second week of treatment. Hunched posture was observed for a single female on Day 10 post-coitum.
Slight salivation was seen on one or multiple days after treatment among animals of the 180 and 540 mg/kg/day dose groups. Taking into account the nature and in general minor severity of the effect and its time of occurrence (i.e., after treatment), this sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights, body weight gain and gravid uterus adjusted body weight gain of test material treated animals remained in the same range as control over the treatment period at 180 mg/kg/day.
At 540 mg/kg/day, mean body weight loss was observed over Days 6-9 post-coitum (-6.4 vs. +12.3 grams in control), followed by normal body weight gain over Days 9-18 post-coitum and a lower body weight gain over Days 18-21 post-coitum (35.0 vs 39.6 grams in control). The combination of body weight loss early during gestation and lower body weight gain towards the end of gestation resulted in a lower overall body weight gain over Days 6-21 post coitum (89.3 vs. 114.4 gram in control) and lower mean body weights between Days 9 21 post-coitum (up to 7% lower than control). Also, body weight gain corrected for gravid uterus weight was lower (12.99 vs. 35.61 gram in control).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No test material-related changes in food consumption were recorded at 180 mg/kg/day.
At 540 mg/kg/day, lower mean food consumption was observed over the whole treatment period (not reaching statistical significance over Days 18-21 post-coitum). Changes compared to control were highest during the first days of treatment (46% lower than control over Days 6-9 post-coitum), followed by partial recovery. Overall mean food consumption was 22% lower than control.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum levels of TSH and T4 were considered to be unaffected by treatment with the test material.
T3 serum levels were considered to be unaffected by treatment with the test material at 60 mg/kg/day.
At 180 and 540 mg/kg/day, mean T3 serum levels were lower compared to control (0.87 and 0.73x of control, respectively). Mean values remained within the central 95% range of the historical control data.

Historical Control Data for pregnant Han Wistar Rats (2020-2022):
T3 (ng/mL); mean (central 95% range): 0.424 (0.270-0.683); n=347

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related changes in thyroid gland weight (absolute and relative to body weight) were observed up to 180 mg/kg/day.
At 540 mg/kg/day, mean absolute thyroid weight was 7% lower compared to control, without reaching statistical significance. This was ascribed to the lower terminal body weight at the high dose (6.7% below control), as mean thyroid weight relative to body weight was comparable to control.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to be related to treatment with the test material up to 180 mg/kg/day.
At 540 mg/kg/day, 2/22 females were observed with a small-sized thymus. As this was observed at the high dose only, a test material-relation could not be excluded.
Other findings that were noted, including those in the thyroid gland were considered to be unrelated to treatment with the test material, as they occurred in control animals only, or in the absence of a dose response.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related microscopic observations in the thyroid glands.
In one female at 60 mg/kg/day, hypertrophy of the follicular cell epithelium was observed at a minimal degree. This was the only recorded microscopic finding and regarded unrelated to treatment in the absence of a dose response and single occurrence at the 60 mg/kg/day.

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The numbers of pre- and postimplantation loss in the control and test material-treated groups were comparable and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The numbers of pregnant females in the control and test material-treated groups were comparable and in the range of normal biological variation. In total, 20/22, 20/22, 17/22 and 21/22 females of the control, 60, 180 and 540 mg/kg/day groups, respectively, were pregnant and therefore available for ovarian and uterine examination at scheduled necropsy.

Key result

Dose descriptor:

NOAEL

Remarks:

materal

Effect level:

180 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean fetal weights (both sexes) were considered to be unaffected by treatment with the test material up to 180 mg/kg/day.
At 540 mg/kg/day, mean fetal weights (male, female and combined) were 2.22, 3.63 and 2.84% lower, respectively, when compared to the control group (only reaching statistical significance for females).

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was considered to be unaffected by treatment with the test material.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test material-related effects in litter size.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and corrected for body weight) in male and female fetuses was considered not to be affected by treatment with the test material.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related external malformations and variations were recorded.
External malformations were observed in two fetuses originating from two litters at 180 mg/kg/day. Fetus No. 53-R10 had a malpositioned eye bulge, cleft lip, meningoencephalocele and absent palatal rugae. Fetus No. 64-L4 presented with a curled tail. As these malformations were each observed only once and in the mid-dose group only, they were considered to be unrelated to treatment with the test material.
External variations or incidental findings were not observed.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related skeletal malformations and variations were recorded.
Skeletal malformations were observed in a single fetus at 60, 180 and 540 mg/kg/day each. Due to the single occurrences of all recorded malformations, they were considered unrelated to treatment with the test material.
The skeletal malformations observed for Fetus No. 53-R10 corresponded to the external malformations noted for this fetus.
Skeletal variations were observed in the forelimb, pelvic girdle, (supernumerary) rib, scapula, skull, sternebra and vertebra. All were observed either infrequently, at instances comparable to the control group, in the absence of a dose response or were limited to a control fetus only. Therefore, they were considered not to be related to treatment with the test material.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related visceral malformations and variations were recorded.
Visceral malformations were observed in a single fetus at 60 mg/kg/day (Fetus No. 41-10). This fetus was observed with a malpositioned testis and epididymis. As this occurred in a single low-dose fetus, these occurrences were considered to be unrelated to the treatment with the test material.
Visceral variations in this study were infrequently observed. They were found in the carotid artery, thoracic wall, liver and ureter. As these variations were observed at low incidences, within the available historical control data and/or in the absence of a dose response, these were considered not related to treatment with the test material.
The incidental findings of discolored liver and spleen, not otherwise classified as malformation or variation, were observed in a single fetus at 540 mg/kg/day and deemed to be a chance discovery.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

> 540 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose without effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

540 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD 414, GLP (RL1)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 414

Time-mated female Wistar Han rats were treated with the substance from Days 6 to 20 post coitum, inclusive, by daily oral gavage at dose levels of 60, 180 or 540 mg/kg bw/day. The rats of the control group received the vehicle, corn oil, alone.

At 540 mg/kg bw/day, tremors and/or generalized twitches, as well as erected fur and hunched posture were observed. Furthermore, body weight loss over Days 6-9 post-coitum, followed by normal body weight gain over Days 9-18 post-coitum and finally a lower body weight gain over Days 18-21 post-coitum and a lower overall food consumption were observed. The combination of effects on body weight gain and food consumption resulted in a lower gravid uterus adjusted body weight gain compared to control. Based on the nature of clinical signs and the magnitude of change of body weight (gain) and food consumption, this was considered to be adverse. At necropsy, a small-sized thymus was observed at a low incidence. As this finding was limited to the high dose group only, a possible relation to treatment with the test material could not be excluded. However, in the absence of histological evaluation of this organ, adversity could not be assessed.

A reduction of T3 was observed at 180 and 540 mg/kg bw/day. As values remained within the available historical control data, this was considered to be non-adverse .

The lower mean absolute thyroid weight at 540 mg/kg bw/day was considered an indirect effect of the lower body weight and therefore non-adverse, as thyroid weight relative to body weight was considered unaffected.

At 540 mg/kg bw/day, lower fetal weights were observed (both sexes). Based on the magnitude of change compared to the control group, this was considered to be non-adverse. It should be noted that the lower fetal weights were observed in the presence of maternal toxicity.

In conclusion, based on the results of this prenatal developmental toxicity study in time mated female Wistar Han rats the following maternal and developmental No Observed Adverse Effect Levels (NOAELs)  were established:

Maternal NOAEL: 180 mg/kg bw/day (based on clinical signs, lower body weight and lower food consumption observed at 540 mg/kg bw/day).

Developmental NOAEL: at least 540 mg/kg bw/day.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on reproduction and developmental toxicity the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.