2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 Jan 2022 to 10 Feb 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 25 Jan 2022 to 10 Feb 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL
- Name: C.I. Pigment Yellow 185
- CAS: 76199-85-4
- Batch identification: 200007P040
- Content: 92.5% (based on 7.5 g/100 g Soxhlet extractables), w (pigment) = 91.7 g/100 g
- Homogeneity: yes
- Storage stability: expiry date 01. Feb. 2030, the stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility
- Physical state: solid/yellow
- Storage conditions: ambient (room temperature)

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany, Sulzfeld (parental animals, all other animals used in this study (F1 generation pups) were derived from the supplier-provided animals)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males 14-15 weeks old, females 13 weeks old
- Weight at study initiation: weight variation did not exceed 20 percent of the mean weight of each sex
- Housing: during pretreatment rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp); during premating (males and females), mating and postmating period (males only) of the study, the rats were housed together (up to 2 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp); during mating, gestation, lactation and after weaning, the females were housed individually in Polycarbonate cages type III with the following exceptions: i) during overnight mating, male and female mating partners were housed together, ii) pregnant animals and their litters were housed together until PND 13 pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation; for enrichment wooden gnawing blocks were added and in Polysulfonate cages large play tunnels 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
- Diet: mouse and rat maintenance diet “GLP”, supplied by Granovit AG, Kaiseraugst, Switzerland, ad libitum
- Water: water bottles, ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 Jan 2022 (supply of animals/beginning of acclimatization) To: 20 Apr 2022 (sacrifice parental females)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% sodium carboxymethyl cellulose suspension in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

- The test substance preparations were prepared in intervals, which took into account the analytical results of the stability verification
- For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water in a calibrated beaker and intensely mixed with a homogenizer
- Before and during administration, the preparations were kept homogeneous with a magnetic stirrer
- Amount of vehicle (if gavage): 10 mL/kg body weight, the calculation of the administration volume was based on the most recent individual body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses were carried out as a separate study under the responsibility of the study director of this test facility and in compliance with GLP.
Analytical verifications of the stability of the test substance in the carrier for a period of 7 days at room temperature were verified prior to the start of the study in a comparable batch.
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. 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.

Stability analysis:
The stability of the test substance in the carrier was demonstrated for a period of 7 days at room temperature

Concentration control and homogeneity analysis:
The results of the analyses of the test substance preparations confirmed the correctness of the prepared concentrations of the low-, mid- and high dose group samples or undercut the limits of the analytical method only marginally. The mean values of most of the samples corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.
The mean values of four samples were found to be slightly below the acceptable limit and ranged between 87.6-89.1% of the nominal concentration of 1.09 g/ 100 mL and 10.90 g/ 100 mL (low- and high-dose concentration, respectively). If all low or high concentration samples were summarized the values were within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.
The values of one sample- from the low-dose concentration and one from the mid-dose concentration showed deviations of more than 20% to the nominal concentrations of 1.09 g/100 mL or 3.27 g/ 100 mL, respectively. However, if all low or mid concentration samples were summarized, the mean values were either still within the limit of the analytical method (97.7%, low-dose concentration) or the departure from the target concentration was only minor (-2.6%, mid-dose concentration).
The homogeneous distribution of the test substance in the vehicle (0.5% CMC in deionized water) could be demonstrated in the first sample set of the low- and high-dose group, but not in the second sample set. However, the preparation of the suspension, i.e. deionized water containing 0.5% CMC and the test item, was stirred until homogeneity was visually given.
It has to be noted that the test substance reveals the characteristics of a pigment which is not soluble in water. Taking these characteristics into account, the detected nominal concentration and homogeneity values can be considered acceptable.

Details on mating procedure:

- M/F ratio per cage:
- Length of cohabitation: overnight in a 1:1 ratio for maximum of 2 weeks, throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group
- Proof of pregnancy: 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 "GD 0" and the following day "GD 1"

Duration of treatment / exposure:

The duration covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as the entire gestation and lactation period in females up to one day prior to the day of scheduled sacrifice of the animals. Females in labor were not treated. The male and female animals were sacrificed after 35 and 64 days, respectively.

Frequency of treatment:

once daily at approximately at the same time in the morning

Duration of test:

The duration covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as the entire gestation and lactation period in females up to one day prior to the day of scheduled sacrifice of the animals. The male and female animals were sacrificed 35 and 64 days, respectively.

Dose / conc.:

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

Remarks:

control group

Dose / conc.:

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

Remarks:

low dose level

Dose / conc.:

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

Remarks:

mid dose level

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

high dose level

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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

DETAILED CLINICAL OBSERVATIONS: Yes
- 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 within 5 hours after the administration
- Abnormalities and changes were documented daily for each animal
- The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams
- 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

BODY WEIGHT: Yes
- Determined once a week at the same time of the day (in the morning) until sacrifice, with the following exceptions: i) 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; ii) females with litter were weighed on the day of parturition (PND 0), 1, 4, 7, 10 and 13
- Females without positive evidence of sperm, females without litter and females after weaning (PND 13) were weighed once a week together with the males

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Once a week for male and female parental animals, with the following exceptions: i) food consumption was not determined after the 2nd premating week (male parental animals); ii) food consumption of the females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14, and 14-20; iii) food consumption of 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

POST-MORTEM EXAMINATIONS: Yes
- All parental animals were sacrificed by decapitation under isoflurane anesthesia, females were sacrificed on day 64 after beginning of administration of test substance
- Gross necropsy: The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs
- Organ weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal 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
All paired organs were weighed together (left and right)
- The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Cervix
3. Coagulating glands
4. Epididymides (modified Davidson’s solution)
5. Ovaries (modified Davidson’s solution)
6. Oviducts
7. Prostate
8. Seminal vesicles
9. Testes (modified Davidson’s solution)
10. Thyroid glands (with parathyroid glands)
11. Uterus
12. Vagina
- Histopathology: Fixation was followed by histotechnical processing, examination by light microscopy and assessment of ovaries, a correlation between gross lesions and histopathological findings was attempted.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Uterus weight: Yes
- Number of implantations: Yes, to determine the number of implantation sites, the apparently non-pregnant uteri were stained for about 5 minutes in 1% ammonium sulfide solution, then the uteri were rinsed carefully with 0.9% NaCl solution, thereafter the implantation sites were recorded for calculation of the postimplantation loss

Blood sampling:

- Blood samples from all parental females at PND 14 at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia
- The parental animals were fastened before blood sampling
- In the samples of the dams at PND 14 no thyroid hormones were measured, because no relevant changes of T4 and TSH values in parental males were observed

Statistics:

See "Any other information on materials and methods incl. tables" for Table 1 for statistics of the clinical examinations, Table 2 for statistics of clinical pathology and Table 3 for statistics of pathology.

Indices:

FEMALE REPRODUCTION AND DELIVERY DATA:

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

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

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

Viability index (%) = (number of live pups on day 4* after birth) / (number of live pups on the day of birth) x 100
* 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)

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

Historical control data:

Please refer to the attachment in section "Overall remarks, attachments".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All animals of the treatment groups 1-3 (100, 300 and 1000 mg/kg bw/d, respectively) showed discolored feces during the entire study period. This finding is clearly related to the test substance but was assessed as being not adverse.
Nine high-, seven mid- and one low-dose male animals showed salivation after dosing during in-life phase. Salivation after dosing was also observed in one high-dose female during in-life and mating phase, as well as in eight high-dose and one mid-dose females during gestation phase. During lactation phase salivation after dosing was noted in all high-dose, five mid- and one low-dose females. It is likely, that these temporary findings were induced by a bad taste of the test substance. It is, however, not considered to be an adverse toxicologically relevant finding (see Attachments for result tables).

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight was decreased in high - and mid-dose females attaining statistical significance on gestation days 7 and 14 (up to -9.8 % and -7.4% versus control group, respectively). During lactation the body weight of the high - dose females was statistically significantly below the concurrent control values on days 1, 4 and 10 ( up to -9.5%) and additionally in mid-dose females during lactation day 4 (-7.6%). The body weight change was statistically significantly below the concurrent control value in high -dose female animals during in-life days 0-7 and gestation days 7-14 (- 265.0% and -56.1%, respectively).
Mean body weights and body weight change of all parental animals in the low-dose group (100 mg/kg bw/d) were comparable to the concurrent control values during the entire study period (see Attachments for result tables).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was statistically significantly decreased in high- and mid-dose females during in-life days 0 - 7 and 0 - 13 (up to -37.4% and -20.6% versus control, respectively). During gestation days 0 – 7, 7 - 14 and 0 - 20 the high dose females consumed up to 17.7% less food compared to control and the mid-dose females showed a decreased food consumption on gestation days 0 – 7, 7 – 14, 14 - 20 and 0 - 20 (up to -15.0% versus control group). Food consumption of the low-dose females was comparable to the concurrent control values during the entire study (see Attachments for result tables).

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

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):

When compared to control group 0 (=100%), the mean relative weights of the uterus were significantly changed in one or more test groups in females (see Table 4 in "Any other information on results incl. tables").
All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The statistically significantly decreased mean relative uterus weights in test groups 1 and 2 were not regarded to be treatment-related as there was no clear dose response and no significant change in test group 3 (see Attachments for result tables).

Gross pathological findings:

no effects observed

Description (incidence and severity):

All findings occurred individually. They were considered to be incidental or spontaneous in
origin and without any relation to treatment.
Fertility: The female animals which were not pregnant as well as the male mating partners did not show relevant gross lesions.

Neuropathological findings:

not examined

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

All findings occurred in the control group. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Fertility: The female animals which were not pregnant as well as the male mating partners did not show relevant histopathological findings. The male mating partner of one female animal (control group) showed an unilateral sperm granuloma in the epididymidis of the left side and an unilateral diffuse severe degeneration of the testicular tubules also on the left side, which was assumed to be the cause for the infertility of this couple.

Other effects:

no effects observed

Description (incidence and severity):

Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter was 90% in the control group and 100% in all test groups 1-3.
The mean duration until sperm was detected (GD 0) varied between 2.2 and 3.4 days, which reflect the normal biological variation.
The fertility index ranged between 88.9% (control group), 90% (test group 2) and 100% (test group 1 and 3) without showing any relation to dosing.
None of the non-pregnant females had any relevant gross or histopathological findings.
The gestation index was 100% in control and test groups 1-3 and the mean duration of gestation varied between 22.5 (control), 22.1 (test group 1), 22.4 (test group 2) and 21.9 days (test group 3).

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

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 (12.6 / 13.7 / 12.8 and 11.7 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 statistically significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (12.4 / 13.6 / 12.3 and 10.8 pups/dam in test groups 0 - 3, respectively).

Other effects:

no effects observed

Description (incidence and severity):

Accordingly, live birth indices were 94.9% / 94.1% / 94.6% and 100% in test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d), respectively. The mean percentage of stillborn pups was 5.1 %/ 5.9% / 5.4 %/ 0.0 %, and the mean percentage of perinatal loss was 5.6% / 7.1% / 4.4% and 0.0% in test groups 0 - 3, respectively.

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights were statistically significantly decreased in male pups of the high-dose group on PND 1-13 (up to -20.8% versus control group), in female pups on PND 4-13 (up to -21.2% versus control group) and also on PND 4-13 if male and female pups were combined (up to -22.7% versus control group).
The mean body weight change was statistically significantly decreased between PND 1 and 13 in male and female pups of test group 3 (up to -23.4% males and -24.5% females versus control group) and also if male and female pups were combined (up to -26.0% versus control group).
The mean pup body weight and pup body weight change values of the low- and mid-dose pups were comparable to the concurrent control values throughout the entire study, except for low dose male pups, which showed a statistically significantly decreased mean body weight change on PND 1-4 (-20.9 % versus control group). This finding was regarded as incidental and not treatment-related since there is no relation to the dose.
One male runt was seen in the control, two female runt was seen in test group 1 (100 mg/kg bw/d) and three male runts were seen each in test group 2 and 3 (300 and 1000 mg/kg bw/d, respectively). This finding reflects the normal range of biological variation inherent in the strain used in this study and was therefore assumed as incidental and not treatment-related (see Attachments for result tables).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The test substance did not influence pup viability and survival in any of the treated groups.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

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. However, the mean percentage of male pups on day 0 was decreased in test group 1 compared to control group (58.9 %/ 46.2** %/ 57.8 %/ 46.4 %). Accordingly, the mean percentage of female pups showed increased values in test group 1 compared to the concurrent control (41.1 %/ 53.8** %/ 42.2 %/ 53.6 % test groups 0-3, respectively). The respective values reflect the normal range of biological variation inherent in the strain used in this study and no relation to the dose occurred. Therefore, this finding was regarded as not related to the treatment.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The anogenital distance and anogenital index of all test substance treated male and female pups were comparable to the concurrent control values.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs:
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
In one low-dose female pup an absent tail was observed and was missing/cannibalized on PND 2.
One mid-dose female pup showed general poor condition, pale skin, reduced nutritional condition and a distended abdomen on PND 7-8 and was missing/cannibalized on PND 9.
One mid-dose male pup showed an injury on PND 4-10 and an absent tail on PND 11-13.The pup was sacrificed on PND 13.
One high-dose female pup showed a palpable mass through the dorsal skin during PND 10-13. The pup was sacrificed on PND 13.
These observations were not considered to be associated with the test compound since only single pups were affected.

Gross pathological findings:
A few pups showed spontaneous findings at gross necropsy, such as an absent tail and post mortem autolysis.
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.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity of F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 4: Relative organ weights in parental animals

Relative weights	Females
Test group (mg/kg bw/day)	1 (100)	2 (300)	3 (1000)
Uterus	-21.7%**	-13.4%*	-16.3%

*: p <= 0.05, **: p <= 0.01

Conclusions:

Under the conditions of the present Reproduction/Developmental Toxicity Screening Test the oral administration of the test substance to male and female Wistar rats resulted in signs of systemic toxicity, such as reduced food consumption, body weights and body weight changes at a concentration of 1000 mg/kg bw/d in both male and female animals. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 300 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 300 mg/kg bw/d, based on lower pup weights at 1000 mg/kg bw/d.

Executive summary:

In an OECD 421 study, the test substance was administered daily to groups of 10 male and 10 female Wistar rats (P0 animals) by gavage at dose levels of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d, test groups 1-3, respectively) to screen for potential systemic, reproductive and developmental toxicity. Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only. 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.

Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was demonstrated over a period of 7 days under ambient conditions.

Regarding clinical examination, high-dose (1000 mg/kg bw/d) parental males showed a reduced food consumption during in-life days 0 - 13 (-21.9% compared to control) resulting in decreased body weights (d 13 - 34, up to -9.7% versus control) and decreased body weight change values (d 0 – 34, up to -214.1% versus control).

In high-dose (1000 mg/kg bw/d) parental females a reduction in food consumption was observed during in-life days 0 - 13 and gestation days 0 - 20 (up to -37.4% and -17.7% versus control, respectively). Their body weights were also reduced on gestation days 7 and 14, as well as on lactation days 1, 4 and 10 (up to -9.8% compared to control). Additionally, the body weight change was below the concurrent control during in-life days 0 – 7 and gestation days 7 – 14 (-265.0% and -56.1%, respectively).

All in all, the complex of decreased food consumption, body weight and body weight change in the parental animals of the high-dose group was regarded as an indication that these local findings became systemically effective and, thus, were considered as treatment-related and adverse.

The decreased body weight change values of the mid-dose males during in-life days 0 - 34 (up to -72.6%) as well as the reduced food consumption in mid-dose females during in-life days 0 - 13 and gestation days 0 – 20 (up to -20.6% and – 15.0% versus control, respectively), which resulted in decreased body weight values on single days during gestation and lactation (up to -7.6% versus control), were considered to be indicative for a beginning systemic toxicity, thus treatment-related, but not yet adverse.

Male and female parental animals in the low-dose group (100 mg/kg bw/d) showed no adverse findings in any of the examined parameters.

Concerning T4 and TSH measurements, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d.

Regarding pathology, the terminal body weight of the parental males of test group 3 (1000 mg/kg bw/d) was significantly decreased (-9.0%), which was regarded as treatment-related and adverse. The statistically significantly decreased mean absolute epididymis weight and the statistically significantly increased mean relative testis weight in this test group were regarded to be secondary to the decreased terminal body weight. All other findings occurred either individually or were biologically equally distributed about control and treatment groups. They were considered to be incidental and spontaneous in origin and without any relation to treatment.

Fertility, reproductive performance and delivery were not impaired by test-substance up to the highest dose level (1000 mg/kg bw/d), as was demonstrated by unchanged fertility, gestation and live birth indices of pups in all test groups.

Regarding developmental toxicity, no adverse findings were observed in terms of pup status, viability and survival. Regarding pup growth, no differences were observed in birth weights but on PND 1-13 high-dose (1000 mg/kg bw/d) F1 males as well as on PND 4-13 high-dose F1 females showed decreased body weights (up to -21.2% compared to control), resulting in a reduction of body weight of males and females combined on PND 4-13 (up to -22.7% compared to control). The mean body weight changes of test group 3 were also decreased on PND 1-13 in male and female pups (up to -23.4% males and -24.5% females versus control group) and also if male and female pups were combined (up to -26.0% versus control group). It should be noted that distinct maternal / parental toxicity was observed (see above). However, independently of the primary or secondary nature of the reduced pup weights the magnitude of the growth disturbance suggests that it is unlikely for the pups to make a short-term and full recovery. Thus, this effect was considered to be treatment-related and adverse.

No test substance-related influence on body weight (change) of the low- and mid-dose male and female F1 pups were noted.

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.

Under the conditions of the present Reproduction/Developmental Toxicity Screening Test the oral administration of the test substance to male and female Wistar rats resulted in signs of systemic toxicity, such as reduced food consumption, body weights and body weight changes at a concentration of 1000 mg/kg bw/d in both male and female animals. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 300 mg/kg bw/d for male and female Wistar rats.

The no observed adverse effect level (NOAEL) for developmental toxicity was 300 mg/kg bw/d, based on lower pup weights at 1000 mg/kg bw/d.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany

Limit test:

no

Test material

Test material information

1

Reference substance name:

2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

EC Number:

278-388-8

EC Name:

2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Cas Number:

76199-85-4

Molecular formula:

C16H11N5O4

IUPAC Name:

2-cyano-N-methyl-2-[3-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)-2,3-dihydro-1H-isoindol-1-ylidene]acetamide

Test material form:

solid: nanoform

Details on test material:

Yellow powder
Batch 200007P040
Storage conditions: ambient (room temperature)
Expiry date: 01-Feb-2030

- Shape of particles: spherical
- Surface area of particles: 46.6 m2/g
- Crystal structure: crystalline
- Coating: none

Specific details on test material used for the study:

TEST MATERIAL
- Name: C.I. Pigment Yellow 185
- CAS: 76199-85-4
- Batch identification: 200007P040
- Content: 92.5% (based on 7.5 g/100 g Soxhlet extractables), w (pigment) = 91.7 g/100 g
- Homogeneity: yes
- Storage stability: expiry date 01. Feb. 2030, the stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility
- Physical state: solid/yellow
- Storage conditions: ambient (room temperature)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

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:

TEST ANIMALS
- Source: Charles River Laboratories, Germany, Sulzfeld (parental animals, all other animals used in this study (F1 generation pups) were derived from the supplier-provided animals)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males 14-15 weeks old, females 13 weeks old
- Weight at study initiation: weight variation did not exceed 20 percent of the mean weight of each sex
- Housing: during pretreatment rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp); during premating (males and females), mating and postmating period (males only) of the study, the rats were housed together (up to 2 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp); during mating, gestation, lactation and after weaning, the females were housed individually in Polycarbonate cages type III with the following exceptions: i) during overnight mating, male and female mating partners were housed together, ii) pregnant animals and their litters were housed together until PND 13 pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation; for enrichment wooden gnawing blocks were added and in Polysulfonate cages large play tunnels 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
- Diet: mouse and rat maintenance diet “GLP”, supplied by Granovit AG, Kaiseraugst, Switzerland, ad libitum
- Water: water bottles, ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 Jan 2022 (supply of animals/beginning of acclimatization) To: 20 Apr 2022 (sacrifice parental females)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% sodium carboxymethyl cellulose suspension in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

- The test substance preparations were prepared in intervals, which took into account the analytical results of the stability verification
- For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water in a calibrated beaker and intensely mixed with a homogenizer
- Before and during administration, the preparations were kept homogeneous with a magnetic stirrer
- Amount of vehicle (if gavage): 10 mL/kg body weight, the calculation of the administration volume was based on the most recent individual body weight

Details on mating procedure:

- M/F ratio per cage:
- Length of cohabitation: overnight in a 1:1 ratio for maximum of 2 weeks, throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group
- Proof of pregnancy: 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 "GD 0" and the following day "GD 1"

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses were carried out as a separate study under the responsibility of the study director of this test facility and in compliance with GLP.
Analytical verifications of the stability of the test substance in the carrier for a period of 7 days at room temperature were verified prior to the start of the study in a comparable batch.
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. 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.

Stability analysis:
The stability of the test substance in the carrier was demonstrated for a period of 7 days at room temperature

Concentration control and homogeneity analysis:
The results of the analyses of the test substance preparations confirmed the correctness of the prepared concentrations of the low-, mid- and high dose group samples or undercut the limits of the analytical method only marginally. The mean values of most of the samples corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.
The mean values of four samples were found to be slightly below the acceptable limit and ranged between 87.6-89.1% of the nominal concentration of 1.09 g/ 100 mL and 10.90 g/ 100 mL (low- and high-dose concentration, respectively). If all low or high concentration samples were summarized the values were within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.
The values of one sample- from the low-dose concentration and one from the mid-dose concentration showed deviations of more than 20% to the nominal concentrations of 1.09 g/100 mL or 3.27 g/ 100 mL, respectively. However, if all low or mid concentration samples were summarized, the mean values were either still within the limit of the analytical method (97.7%, low-dose concentration) or the departure from the target concentration was only minor (-2.6%, mid-dose concentration).
The homogeneous distribution of the test substance in the vehicle (0.5% CMC in deionized water) could be demonstrated in the first sample set of the low- and high-dose group, but not in the second sample set. However, the preparation of the suspension, i.e. deionized water containing 0.5% CMC and the test item, was stirred until homogeneity was visually given.
It has to be noted that the test substance reveals the characteristics of a pigment which is not soluble in water. Taking these characteristics into account, the detected nominal concentration and homogeneity values can be considered acceptable.

Duration of treatment / exposure:

The duration covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as the entire gestation and lactation period in females up to one day prior to the day of scheduled sacrifice of the animals. Females in labor were not treated. The male and female animals were sacrificed after 35 and 64 days, respectively.

Frequency of treatment:

once daily at approximately at the same time in the morning

Details on study schedule:

- 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
- Males and females from the same dose group were mated after two weeks of treatment, overnight in a ratio of 1:1
- The females were allowed to deliver and rear their pups until PND 4 (standardization) or PND 13

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

control group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

low dose level

Doses / concentrations 3

Dose / conc.:

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

Remarks:

mid dose level

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

high dose level

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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

DETAILED CLINICAL OBSERVATIONS: Yes
- 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 within 5 hours after the administration
- Abnormalities and changes were documented daily for each animal
- The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams
- 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

BODY WEIGHT: Yes
- Determined once a week at the same time of the day (in the morning) until sacrifice, with the following exceptions: i) 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; ii) females with litter were weighed on the day of parturition (PND 0), 1, 4, 7, 10 and 13
- Females without positive evidence of sperm, females without litter and females after weaning (PND 13) were weighed once a week together with the males

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Once a week for male and female parental animals, with the following exceptions: i) food consumption was not determined after the 2nd premating week (male parental animals); ii) food consumption of the females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14, and 14-20; iii) food consumption of 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

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 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 parental female with scheduled sacrifice.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible)
- 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 less than 8 pups

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number of pups, sex, number of liveborn and stillborn pups, postnatal mortality, presence of gross anomalies, body weight change, anogenital distance (AGD; anogenital index = anogenital distance [mm] / cubic root of pup weight [g]), nipple/areola anlagen in male pups

GROSS EXAMINATION OF DEAD PUPS: Yes
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 by decapitation. 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.


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

CLINICAL PATHOLOGY (THYROID HORMONES)
- Blood samples from all parental females at PND 14 and all parental males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia
- The parental animals were fastened before blood sampling
- Blood samples from the adult males were assessed for serum levels for thyroid hormones T4 and TSH
- In the samples of the dams at PND 14 no thyroid hormones were measured, because no relevant changes of T4 and TSH values in parental males were observed
- For the same reason no T3 values were measured in any sample collected for thyroid hormone measurement
- All generated serum samples were frozen at -80°C until measurement
- For parameters and methods used to determine TSH and T4 concentrations, refer to Table 2 in "Any other information on materials and methods incl. tables"

SACRIFICE
- All parental animals were sacrificed by decapitation under isoflurane anesthesia

GROSS NECROPSY
- The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs

HISTOPATHOLOGY / ORGAN WEIGHTS
Weight parameters:
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal 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
All paired organs were weighed together (left and right)

Organ/tissue fixation:
The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Cervix
3. Coagulating glands
4. Epididymides (modified Davidson’s solution)
5. Ovaries (modified Davidson’s solution)
6. Oviducts
7. Prostate
8. Seminal vesicles
9. Testes (modified Davidson’s solution)
10. Thyroid glands (with parathyroid glands)
11. Uterus
12. Vagina

Histopathology:
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the Table 4 in "Any other information on materials and methods incl. tables". Special emphasis was given to the stages of spermatogenesis and histopathology of interstitial testicular cell structure. A correlation between gross lesions and histopathological findings was attempted.

Statistics:

See "Any other information on materials and methods incl. tables" for Table 1 for statistics of the clinical examinations, Table 3 for statistics of clinical pathology and Table 5 for statistics of pathology.

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 parental breeding pairs.
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:

Male mating index (%) = (number of males with cofirmed mating *) / (number of males placed with females) x 100
* defined by a female with vaginal sperm or with implants in utero


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 was detected and gestational status was recorded for parental females.
For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:

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


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

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

Offspring viability indices:

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 was 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) x 100
* 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)

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All male and female animals of the treatment groups 1-3 (100, 300 and 1000 mg/kg bw/d, respectively) showed discolored feces during the entire study period. This finding is clearly related to the test substance but was assessed as being not adverse.
Nine high-, seven mid- and one low-dose male animals showed salivation after dosing during in-life phase. Salivation after dosing was also observed in one high-dose female during in-life and mating phase, as well as in eight high-dose and one mid-dose females during gestation phase. During lactation phase salivation after dosing was noted in all high-dose, five mid- and one low-dose females. It is likely, that these temporary findings were induced by a bad taste of the test substance. It is, however, not considered to be an adverse toxicologically relevant finding (see Attachments for result tables).

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of the high-dose males was statistically significantly below the concurrent control values during in-life days 13-34 ( up to - 9.7 %) resulting in decreased body weight change values during in-life days 0-13, 28-34 and 0-34 (up to - 214.1% versus control) . The body weight change values of the mid-dose males were also statistically significantly below the current control values during in-life days 0-7 and 0-34 (- 72.6% and - 29.5%, respectively).
The body weight was decreased in high - and mid - dose females attaining statistical significance on gestation days 7 and 14 (up to - 9.8 % and - 7.4% versus control group, respectively). During lactation the body weight of the high - dose females was statistically significantly below the concurrent control values on days 1, 4 and 10 ( up to - 9.5%) and additionally in mid-dose females during lactation day 4 (- 7.6%). The body weight change was statistically significantly below the concurrent control value in high - dose female animals during in-life days 0-7 and gestation days 7-14 ( - 265.0% and - 56.1% , respectively).
Mean body weights and body weight change of all male and female parental animals in the low-dose group (100 mg/kg bw/d) were comparable to the concurrent control values during the entire study period (see Attachments for result tables).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the high-dose males was statistically significantly below the concurrent control values during in-life days 7 – 13 (-18.5%) resulting in decreased values on in-life days 0 – 13 (-21.9% compared to control). Food consumption was also statistically significantly decreased in high- and mid-dose females during in-life days 0 - 7 and 0 - 13 (up to -37.4% and -20.6% versus control, respectively). During gestation days 0 – 7, 7 - 14 and 0 - 20 the high dose females consumed up to 17.7% less food compared to control and the mid-dose females showed a decreased food consumption on gestation days 0 – 7, 7 – 14, 14 - 20 and 0 - 20 (up to -15.0% versus control group). Food consumption of the low-dose females was comparable to the concurrent control values during the entire study (see Attachments for result tables).

Food efficiency:

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):

At the end of the administration period, in F0 males of test group 03 (1000 mg/kg bw/d) T4 values were significantly decreased. However, T4 values as well as TSH values were within historical control ranges (F0 males, T4 40.44-72.39 nmol/L, TSH 3.49-10.31 μg/L). No weight change of the thyroids among these individuals was observed. Therefore, the T4 decrease in males of test group 3 was regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

All findings occurred in the control group. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Fertility: The female animals which were not pregnant as well as the male mating partners did not show relevant histopathological findings. The male mating partner of one female animal (control group) showed an unilateral sperm granuloma in the epididymidis of the left side and an unilateral diffuse severe degeneration of the testicular tubules also on the left side, which was assumed to be the cause for the infertility of this couple.

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 test groups 0 - 3. The mean estrous cycle duration and number of cycles was comparable (4.0 / 3.9 / 3.9 and 3.7 days in test groups 0 - 3, respectively).

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter was 90% in the control group and 100% in all test groups 1-3.
The mean duration until sperm was detected (GD 0) varied between 2.2 and 3.4 days, which reflect the normal biological variation.
The fertility index ranged between 88.9% (control group), 90% (test group 2) and 100% (test group 1 and 3) without showing any relation to dosing.
None of the non-pregnant females had any relevant gross or histopathological findings.
The gestation index was 100% in control and test groups 1-3 and the mean duration of gestation varied between 22.5 (control), 22.1 (test group 1), 22.4 (test group 2) and 21.9 days (test group 3).
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 (12.6 / 13.7 / 12.8 and 11.7 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 statistically significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (12.4 / 13.6 / 12.3 and 10.8 pups/dam in test groups 0 - 3, respectively). Accordingly, live birth indices were 94.9% / 94.1% / 94.6% and 100% in test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d), respectively. The mean percentage of stillborn pups was 5.1 %/ 5.9% / 5.4 %/ 0.0 %, and the mean percentage of perinatal loss was 5.6% / 7.1% / 4.4% and 0.0% in test groups 0 - 3, respectively.

Male reproduction data:
For all parental males, which were placed with females to generate F1 pups, copulation was confirmed. The male mating index was 90% in test group 0 (control group) and 100% in test groups 1 – 3 (100, 300 and 1000 mg/kg bw/d).
Fertility was proven for most of the parental males within the scheduled mating interval for F1 litter. Two control and one mid-dose animal did not generate pregnancy. Thus, the male fertility index ranged between 80% (test group 0), 90% (test group 2) and 100% (test group 1 and 3) without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
The male mating partner of one female animal (control group) showed left sided an unilateral 2-5 mm yellow focus and a sperm granuloma in the epididymidis, an enlarged testis and an unilateral diffuse severe degeneration of the testicular tubules. These findings were considered to be the cause for the infertility of this couple.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

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.
In one low-dose female pup an absent tail was observed and was missing/cannibalized on PND 2.
One mid-dose female pup showed general poor condition, pale skin, reduced nutritional condition and a distended abdomen on PND 7-8 and was missing/cannibalized on PND 9.
One mid-dose male pup showed an injury on PND 4-10 and an absent tail on PND 11-13.The pup was sacrificed on PND 13.
One high-dose female pup showed a palpable mass through the dorsal skin during PND 10-13. The pup was sacrificed on PND 13.
These observations were not considered to be associated with the test compound since only single pups were affected.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The test substance did not influence pup viability and survival in any of the treated groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights were statistically significantly decreased in male pups of the high-dose group on PND 1-13 (up to -20.8% versus control group), in female pups on PND 4-13 (up to -21.2% versus control group) and also on PND 4-13 if male and female pups were combined (up to -22.7% versus control group).
The mean body weight change was statistically significantly decreased between PND 1 and 13 in male and female pups of test group 3 (up to -23.4% males and -24.5% females versus control group) and also if male and female pups were combined (up to -26.0% versus control group).
The mean pup body weight and pup body weight change values of the low- and mid-dose pups were comparable to the concurrent control values throughout the entire study, except for low dose male pups, which showed a statistically significantly decreased mean body weight change on PND 1-4 (-20.9 % versus control group). This finding was regarded as incidental and not treatment-related since there is no relation to the dose.
One male runt was seen in the control, two female runt was seen in test group 1 (100 mg/kg bw/d) and three male runts were seen each in test group 2 and 3 (300 and 1000 mg/kg bw/d, respectively). This finding reflects the normal range of biological variation inherent in the strain used in this study and was therefore assumed as incidental and not treatment-related (see Attachments for result tables).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

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 were 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.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few pups showed spontaneous findings at gross necropsy, such as an absent tail and post mortem autolysis.
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.

Histopathological findings:

not examined

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. However, the mean percentage of male pups on day 0 was decreased in test group 1 compared to control group (58.9 %/ 46.2** %/ 57.8 %/ 46.4 %). Accordingly, the mean percentage of female pups showed increased values in test group 1 compared to the concurrent control (41.1 %/ 53.8** %/ 42.2 %/ 53.6 % test groups 0-3, respectively). The respective values reflect the normal range of biological variation inherent in the strain used in this study and no relation to the dose occurred. Therefore, this finding was regarded as not related to the treatment.

Thyroid hormones:
In male and female pups at PND13 (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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 6: Absolute organ weights in parental animals

Absolute weights	Males
Test group (mg/kg bw/day)	1 (100)	2 (300)	3 (1000)
Terminal body weight	-1.9%	-2.3%	-9.0%**
Epididymides	-2.8%	-6.6%*	-8.4%**

*: p <= 0.05, **: p <= 0.01

 

 

Table 7: Relative organ weights in parental animals

Relative weights	Males			Females
Test group (mg/kg bw/day)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Testes	+3.8%	+1.8%	+13.3%*
Uterus				-21.7%**	-13.4%*	-16.3%

*: p <= 0.05, **: p <= 0.01

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present Reproduction/Developmental Toxicity Screening Test the oral administration of the test substance to male and female Wistar rats resulted in signs of systemic toxicity, such as reduced food consumption, body weights and body weight changes at a concentration of 1000 mg/kg bw/d in both male and female animals. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 300 mg/kg bw/d for male and female Wistar rats. The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 300 mg/kg bw/d, based on lower pup weights at 1000 mg/kg bw/d.

Executive summary:

In an OECD 421 study, the test substance was administered daily to groups of 10 male and 10 female Wistar rats (P0 animals) by gavage at dose levels of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d, test groups 1-3, respectively) to screen for potential systemic, reproductive and developmental toxicity. Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only. 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.

Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was demonstrated over a period of 7 days under ambient conditions.

Regarding clinical examination, high-dose (1000 mg/kg bw/d) parental males showed a reduced food consumption during in-life days 0 - 13 (-21.9% compared to control) resulting in decreased body weights (d 13 - 34, up to -9.7% versus control) and decreased body weight change values (d 0 – 34, up to -214.1% versus control).

In high-dose (1000 mg/kg bw/d) parental females a reduction in food consumption was observed during in-life days 0 - 13 and gestation days 0 - 20 (up to -37.4% and -17.7% versus control, respectively). Their body weights were also reduced on gestation days 7 and 14, as well as on lactation days 1, 4 and 10 (up to -9.8% compared to control). Additionally, the body weight change was below the concurrent control during in-life days 0 – 7 and gestation days 7 – 14 (-265.0% and -56.1%, respectively).

All in all, the complex of decreased food consumption, body weight and body weight change in the parental animals of the high-dose group was regarded as an indication that these local findings became systemically effective and, thus, were considered as treatment-related and adverse.

The decreased body weight change values of the mid-dose males during in-life days 0 - 34 (up to -72.6%) as well as the reduced food consumption in mid-dose females during in-life days 0 - 13 and gestation days 0 – 20 (up to -20.6% and – 15.0% versus control, respectively), which resulted in decreased body weight values on single days during gestation and lactation (up to -7.6% versus control), were considered to be indicative for a beginning systemic toxicity, thus treatment-related, but not yet adverse.

Male and female parental animals in the low-dose group (100 mg/kg bw/d) showed no adverse findings in any of the examined parameters.

Concerning T4 and TSH measurements, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d.

Regarding pathology, the terminal body weight of the parental males of test group 3 (1000 mg/kg bw/d) was significantly decreased (-9.0%), which was regarded as treatment-related and adverse. The statistically significantly decreased mean absolute epididymis weight and the statistically significantly increased mean relative testis weight in this test group were regarded to be secondary to the decreased terminal body weight. All other findings occurred either individually or were biologically equally distributed about control and treatment groups. They were considered to be incidental and spontaneous in origin and without any relation to treatment.

Fertility, reproductive performance and delivery were not impaired by test-substance up to the highest dose level (1000 mg/kg bw/d), as was demonstrated by unchanged fertility, gestation and live birth indices of pups in all test groups.

Regarding developmental toxicity, no adverse findings were observed in terms of pup status, viability and survival. Regarding pup growth, no differences were observed in birth weights but on PND 1-13 high-dose (1000 mg/kg bw/d) F1 males as well as on PND 4-13 high-dose F1 females showed decreased body weights (up to -21.2% compared to control), resulting in a reduction of body weight of males and females combined on PND 4-13 (up to -22.7% compared to control). The mean body weight changes of test group 3 were also decreased on PND 1-13 in male and female pups (up to -23.4% males and -24.5% females versus control group) and also if male and female pups were combined (up to -26.0% versus control group). It should be noted that distinct maternal / parental toxicity was observed (see above). However, independently of the primary or secondary nature of the reduced pup weights the magnitude of the growth disturbance suggests that it is unlikely for the pups to make a short-term and full recovery. Thus, this effect was considered to be treatment-related and adverse.

No test substance-related influence on body weight (change) of the low- and mid-dose male and female F1 pups were noted.

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.

 

Under the conditions of the present Reproduction/Developmental Toxicity Screening Test the oral administration of the test substance to male and female Wistar rats resulted in signs of systemic toxicity, such as reduced food consumption, body weights and body weight changes at a concentration of 1000 mg/kg bw/d in both male and female animals. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 300 mg/kg bw/d for male and female Wistar rats.

The no observed adverse effect level (NOAEL) for fertility and reproductive performance was 1000 mg/kg bw/d for male and female Wistar rats.

The no observed adverse effect level (NOAEL) for developmental toxicity was 300 mg/kg bw/d, based on lower pup weights at 1000 mg/kg bw/d.