Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.

REACH

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 | CAS number: 76199-85-4

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Key: rat, oral: NOAEL (general systemic toxicity) = 300 mg/kg bw/day, based on reduced food consumption, body weights and body weight changes at 1000 mg/kg bw/day in both sexes; NOAEL (fertility) = 1000 mg/kg bw/day, no adverse effects observed; NOAEL (developmental toxicity) = 300 mg/kg bw/day, based on lower pup weights at 1000 mg/kg bw/day (GLP, OECD 421, 2023)

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

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:

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 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)

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

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:

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

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

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: 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.

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

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

Critical effects observed:

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

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

Critical effects observed:

Reproductive effects observed:

not specified

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

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.

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.

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Study duration:: subacute
Species:: rat

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

Key study: Reproduction/Developmental Toxicity Screening Test

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 (BASF SE, 2023). 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.

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.

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

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.

Effects on developmental toxicity

Description of key information

Key: rat, oral: NOAEL (maternal toxicity) = 300 mg/kg bw/day, based on reduction in food consumption, a decrease in (corrected) body weight gain and changes in clinical pathology parameters (e.g. increased total bilirubin values) at the highest dose level of 1000 mg/kg bw/d; NOAEL (prenatal development) = 1000 mg/kg bw/day, minor effects in the presence of maternal toxicity were indicating a temporary developmental delay and are not adverse (GLP, OECD 414, 2023)

Sup: rat, oral: NOAEL (general systemic toxicity) = 300 mg/kg bw/day, based on reduced food consumption, body weights and body weight changes at 1000 mg/kg bw/day in both sexes; NOAEL (fertility) = 1000 mg/kg bw/day, no adverse effects observed; NOAEL (developmental toxicity) = 300 mg/kg bw/day, based on lower pup weights at 1000 mg/kg bw/day (GLP, OECD 421, 2023)

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 24 Apr 2022 to 22 May 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Limit test:

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- CAS No.: 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: Given
- 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/ appearance: Solid/ yellow
- Storage conditions: 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, Research Models and Services, Germany GmbH
- Age at study initiation: 11-13 weeks
- Weight at study initiation: body weight of the pregnant animals on day 0 varied between 162.6 – 221.2 g
- Housing: During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²). Individual housing is preferred over group housing as the close individual monitoring of food and water consumption as well as of clinical signs of toxicity in pregnant animals is crucial during this period of increased sensitivity. In addition, the control for signs of abortion or fetal loss can only be done in a reliable fashion with single-housed animals. Dust-free wooden bedding was used in this study. As nesting material, compacted fibers of softwood was offered toward the end of gestation in all pregnant females. For enrichment, wooden gnawing blocks and play tunnel were offered. The walls and the floor were cleaned once a week with water containing an appropriate disinfectant.
- Diet: ad libitum, mouse and rat maintenance diet “GLP”, meal, supplied by Granovit AG, Kaiseraugst, Switzerland
- Water: ad libitum, potable tap water in water bottles
- Acclimation period: 1 week

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

IN-LIFE DATES: From: 26-28 Apr 2022 (supply of animals and beginning of acclimatization of the three cohorts) To: 16-18 May 2022 (sacrifice of the three cohorts)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC suspension in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

- Rate of preparation of test substance: The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with vehicle in a calibrated beaker and intensely mixed with a homogenizer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.
- Concentration in vehicle: 0, 1, 3, 10 g/100 mL, respectively
- Amount of vehicle (if gavage): standard dose volume of 10 mL/kg body weight was used for each test group, 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 analytical investigations of the test substance preparations were carried out as a separate study.
Samples of the test substance preparations were sent once (at the beginning of administration) to the analytical laboratory for verification of the concentrations. The samples were also used to verify the homogeneity of the low- and high-concentrations (100 and 1000 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.

Stability: The stability of the test substance in 0.5% CMC suspension in deionized water over a period of 7 days at room temperature had been verified prior to the start of the study with the same batch.

Homogeneity analyses of the test substance preparations: The homogeneous distribution of the test substance in the vehicle (0.5% CMC suspension in deionized water) was demonstrated.

Concentration control analyses of the test substance preparations: The results of the analyses of the test substance preparations confirmed the correctness of the prepared concentrations. The measured concentrations of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant: Animals were paired by the breeder ans supplied on GD 0 (= detection of vaginal plug/sperm). The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”. The animals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6).

Duration of treatment / exposure:

The test substance preparations were administered to the animals from implantation to one day prior to the expected day of parturition (GD 6 to GD 19). The animals of the control group were treated with the vehicle (0.5% CMC suspension in deionized water) in the same way.

Frequency of treatment:

daily, always at approximately the same time in the morning

Duration of test:

The test substance preparations were administered to the animals from implantation to one day prior to the expected day of parturition (GD 6 to GD 19). On GD 20, blood samples were obtained in a randomized order from all females by retrobulbar venous puncture. After blood sampling all surviving dams were sacrificed and examined and fetuses were removed from the uterus and investigated.

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:

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: a check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily before and after treatment period (GD 0-5 and 20), during treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and between 2 and 5 hours after administration

BODY WEIGHT: Yes
- Time schedule for examinations: all animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20
- The body weight change of the animals was calculated based on the obtained results

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 under isoflurane anesthesia by decapitation, in randomized order
- Organs examined: After the dams had been sacrificed, they were necropsied and assessed for gross pathology, special attention being given to the reproductive organs
- The following organ weights were determined in all dams sacrificed on schedule: thyroid glands (with parathyroid glands) (fixed), all paired organs were weighed together (left and right)
- The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution from all dams, sacrificed on schedule: thyroid glands (with parathyroid glands)
- Fixation of thyroid glands from all dams was followed by histotechnical processing (H&E staining) and examination by light microscopy

OTHER:
Corrected (net) body weight gain: the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes, on GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order
The uteri and the ovaries were removed and the following data were recorded:
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes, classified as live fetuses and dead implantations
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters (except of gross pathology including organ sampling and weights) were conducted by technicians unaware of treatment group in order to minimize bias.

Blood sampling:

Blood samples were taken from all females by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood and serum examinations were carried out in a randomized sequence. The parameters depicted in table 1 in "Any other information on materials and methods incl. tables" were determined with EDTA-K3 as anticoagulant using a particle counter. An automatic analyzer was used to examine the clinicochemical parameters (see table 2). The concentrations of TSH were determined by radioimmunassay (RIA), using commercially available RIA test kits and a Gamma-Counter (table 3).

Fetal examinations:

- External examinations: Yes: At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.
- Soft tissue examinations: Yes: The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.
- Skeletal examinations: Yes: The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.
- Anogenital distance of all live rodent pups: Yes, anogenital index = (anogenital distance [mm]) / (cubic root of fetal weight [g])

Statistics:

Please refer to tables 4-6 in "Any other information on materials and methods incl. tables".

Indices:

Conception rate (in %) = (number of pregnant animals) / (number of fertilized animals) x 100

Preimplantation loss (in %) = (number of corpora lutea – number of implantations) / (number of corpora lutea) x 100

Postimplantation loss (in %) = (number of implantations – number of live fetuses) / (number of implantations) x 100

Historical control data:

Please see the attachments in "Overall remarks, attachments".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All females of the test substance treated groups (test group 1-3; 100, 300 or 1000 mg/kg bw/d) showed yellowish discolored feces during the treatment period until terminal sacrifice (GD 20). It was initially observed on GD 7 in the mid- and high-dose groups and on GD 8 in the low-dose group. This feces discoloration mirrors the presence of the yellowish test substance in the gastrointestinal tract but was assessed as of no toxicological relevance.
Piloerection was recorded for one mid-dose female (300 mg/kg bw/d) on GD 19 and 20, and for all high-dose females (1000 mg/kg bw/d) from GD 10 onwards until terminal sacrifice.
Additionally, for one high-dose female (1000 mg/kg bw/d) hypothermia (after treatment) was recorded on GD 10-12.
One low-dose female (100 mg/kg bw/d) had a palpable mass in the mammary line on GD 18-20. Gross pathological examination revealed an enlarged lymph node in the abdominal cavity (see gross pathology findings). Since this was an isolated event in one single dam, it was assessed as incidental and not treatment-related and adverse.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during the entire study period.

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 100, 300 or 1000 mg/kg bw/d).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Only pregnant dams were used for the calculations of body weight and body weight change.
As a consequence of the lower food consumption, the mean body weights (BW; table 7 in "Any other information on results incl. tables") of the high-dose dams (1000 mg/kg bw/d) were statistically significantly lower from GD 8 onwards until scheduled sacrifice on GD 20 (up to 10% below the concurrent control group). The body weight change (BWC; table 8) of the high-dose dams was also distinctly lower at the beginning of the treatment period, attaining statistical significance on GD 6-13 (on GD 6-8, the dams even lost weight (- 2.3 g** [p≤0.01] vs. 6.6 g in control). If calculated for the entire treatment period (GD 6-19) or study period (GD 0-20), the high-dose dams gained about 35% or 24% less weight than the controls (attaining statistical significance). The mean BW of the mid-dose dams (300 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period. However, the BWC of the dams in test group 2 was statistically significantly reduced during GD 6-8 (about 80% below controls) but recovered afterwards. If calculated for the entire treatment period (GD 6-19), the mid-dose dams gained a comparable weight to the controls. The mean BW and the average BWC of the low-dose dams (100 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
Only pregnant dams with scheduled sacrifice (GD 20) were used for the calculation of corrected (net) body weight gain.
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) was statistically significantly lower in test group 3 (1000 mg/kg bw/d - about 69% below control). Furthermore, the carcass weight of the high-dose dams was statistically significantly reduced in comparison to the control group (about 10% below controls).
The corrected body weight gain of test groups 1 and 2 (100 and 300 mg/kg bw/d) revealed no differences of any biological relevance to the corresponding control group. Moreover, mean carcass weights of test groups 1 and 2 remained unaffected by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Only pregnant dams were used for the calculations of mean maternal food consumption.
In test group 3 (1000 mg/kg bw/d), the mean food consumption was statistically significantly lower during the entire treatment period (up to -42% (GD 8-10) below control). If calculated for GD 6-19, the high-dose dams consumed about 29% less food than the controls. The mean food consumption in test group 2 (300 mg/kg bw/d) was statistically significantly lower on GD 6-13 (up to 16% below control) but recovered afterwards and even exceeded the control value at the end of the treatment period. If calculated for the entire treatment period (GD 6-19), the mid-dose dams consumed about 8% less food than the controls. The mean food consumption of the dams in test group 1 (100 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. At gestation day 20 in dams of test groups 2 and 3 (300 and 1000 mg/kg bw/d) absolute reticulocyte and platelet counts were significantly increased. Absolute reticulocyte counts were already significantly increased in dams of test group 1 (100 mg/kg bw/d). However, the values of both parameters were within historical control ranges (dams, absolute reticulocytes 115.3- 181.0 Giga/L, platelets 706.917 Giga/L). Therefore, these alterations were regarded as incidental and not treatment-related. In dams of test group 3 (1000 mg/kg bw/d) absolute and relative lymphocyte and absolute large unstained cell (LUC) counts were significantly increased. Consistently to the higher relative lymphocyte counts, relative neutrophil counts were significantly decreased. Absolute lymphocyte and neutrophil counts in dams of test group 3 were within historical control ranges whereas absolute LUC counts were slightly above this range (dams, absolute lymphocytes 2.58-4.35 Giga/L, absolute neutrophils 1.50-2.23 Giga/L, absolute LUC 0.01-0.02 Giga/L). However, relative LUC counts were so small (0.5%) that the chance to further evaluate these cells in a manual differential blood cell counts (1 of 200 cells) was very low. Therefore, the alterations of lymphocyte and neutrophil counts were regarded as incidental and not treatment-related, whereas the increase of absolute LUC counts was assessed as non-adverse, if at all treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At gestation day 20 in dams of test group 3 (1000 mg/kg bw/d) total bilirubin values were significantly increased whereas total protein, albumin and globulin values were significantly decreased. These alterations were regarded as treatment-related and adverse.
Total protein, albumin and globulin values were already significantly lower in dams of test group 2 (300 mg/kg bw/d) compared to study controls. Albumin values were within the historical control range whereas total protein and globulin values were slightly below their ranges (dams, total protein 56.50-63.74 g/L, albumin 31.14-35.55 g/L, globulins 24.39-29.68 g/L). In fact, the total protein level decrease was based on lower globulin values, because they were the sum of globulins and albumin. Therefore, only one liver parameter, i.e. globulins, was changed in dams of test group 2. Therefore, this change was regarded as treatment-related but not adverse.
The following significant changes were regarded as incidental and not treatment-related because the values were within historical control ranges: increased alanine aminotransferase (ALT) activities and cholesterol values in dams of test group 3 (1000 mg/kg bw/d); increased inorganic phosphate levels in dams of test groups 2 and 3 (300 and 1000 mg/kg bw/d; dams ALT 0.72-1.38 μkat/L, cholesterol 1.82-2.43 mmol/L, inorganic phosphate 1.21-1.99 mmol/L). In dams of test groups 1 and 3 (100 and 1000 mg/kg bw/d) triglyceride levels were significantly decreased, but the change was not dose dependent. Therefore, the mentioned alterations in this section were regarded as incidental and not treatment-related.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

In dams of test group 3 (1000 mg/kg bw/d) T3 and T4 levels were significantly decreased. However, the values of both parameters were within historical control ranges (dams, T3 0.56- 1.31 nmol/L, T4 23.17-38.52 nmol/L) whereas at least T4 values in study controls were in the upper part of this range. Neither any thyroid weight change nor any relevant histologic finding in the thyroid gland were observed. Therefore, T3 and T4 decreases in dams of test group 3 were 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:

no effects observed

Description (incidence and severity):

Only pregnant dams with scheduled sacrifice (GD 20) were used for the calculation of mean gravid uterine weights and mean organ weights.
The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
Absolute weights: All mean absolute weight parameters did not show significant differences when compared to the control group 0.
Relative organ weights: All mean relative weight parameters did not show significant differences when compared to the control group 0.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A yellowish discolored content of the stomach as well as of the entire intestine was recorded in 7 (out of 25) low-dose females and in all mid- and high-dose females, respectively. These discolorations mirrored the presence of the test substance in the gastrointestinal tract. They were considered to be non-adverse, causing no toxic effects by themselves.
Additionally, three spontaneous findings were noted in individual females of test groups 0, 1 and 2 (0, 100 and 300 mg/kg bw/d). An empty stomach was seen in control female, while one low-dose female had an enlarged lymph node at the abdominal cavity. A diaphragmatic hernia was recorded for one mid-dose female. Since these findings occurred in single females without a dose-response relationship, they were assessed as incidental and not treatment-related.
No gross lesions were observed in any of the test groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In test group 1, a minimal increase in the incidence of altered colloid was noted in the thyroid glands. As no organ weight changes were present and no dose-response relationship was observed, this finding was considered incidental and not treatment-related.
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.

Details on maternal toxic effects:

Reproduction data:
Only pregnant dams with scheduled sacrifice (GD 20) were used for the summary of reproduction data.
The conception rate was 92% in test groups 0 and 3 (0 and 1000 mg/kg bw/d) and 100% in test groups 1 and 2 (100 and 300 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study.
There were no test substance-related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences were considered to reflect the normal range of fluctuations for animals of this strain and age.
A 100% postimplantation loss was observed in high-dose female (1000 mg/kg bw/d). Twelve early resorptions were recorded. Complete litter loss in single animals is a common finding and since only one dam was affected, this isolated finding was regarded as incidental and without relation to the treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean fetal weight of test group 3 was statistically significantly lower in comparison to the concurrent control group (3.1 g** vs. 3.6 g in control, all viable fetuses). However, the highdose value was well within the historical control range (HCD both sexes: mean 3.6 g [2.4 – 4.3]). The observed reduced fetal body weights of the high-dose group (1000 mg/kg bw/d) corresponded to a delay in ossification as indicated by a broad range of skeletal variations (see below). The findings in the high-dose fetuses occurred in presence of maternal toxicity and were in the same order of magnitude as reduced food consumption and body weight gain of the dams. Therefore, they were assessed as treatment-related but not as adverse.
The mean fetal weights of test groups 1 and 2 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses.

Anogenital distance of all rodent fetuses:

effects observed, treatment-related

Description (incidence and severity):

The anogenital distance of the male fetuses of test group 3 (1000 mg/kg bw/d) was reduced in comparison to the concurrent control group (-19%; attaining statistical significance) and outside the historical control range (HCD ag distance: mean 2.8 mm [2.3 – 3.4]).
Furthermore, the anogenital index of the male fetuses in test group 3 was statistically significantly reduced (mean 1.50, 14% below control). However, this value was within the historical control range (HCD ag index: mean 1.85 [1.46 – 2.17]).
The anogenital distance and anogenital index in the female fetuses in test group 3 as well as in all male and female fetuses in test groups 1 and 2 were comparable to the concurrent control values.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal external malformations:
An external malformation was recorded in one female high-dose fetus. This fetus had an associated soft tissue malformation. Since this was an isolated event and ‘anasarca’ can be found in the historical control data at comparable incidences, it was assessed as neither treatment-related nor adverse. The overall incidences (tables 9 and 10) of external malformations were comparable to those found in the historical control data.

For fetal external variations and external unclassified observations, see result chapter "Other effects".

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal skeletal malformations:
Skeletal malformations were detected in one fetus of the low-dose group, three fetuses of the mid-dose group and in two fetuses of the high-dose group, as shown in tables 15 and 16.
‘Misshapen basisphenoid’ occurred in one single fetus of test group 1 and can be found in the historical control data, thus, it was assessed as incidental.
The findings ‘shortened scapula’ (mean% 0.0/0.0/1.3/1.4 ) and ‘shortened humerus’ (mean% 0.0/0.0/0.7/0.8) occurred in single fetuses of test groups 2 and 3, twice as combination in one fetus. Both findings can be found in the historical control data at comparable incidences, thus, it was regarded to be incidental and not treatment-related.
The incidence of ‘bent rib’ (mean% 0.0/0.0/2.0*/0.0 [p  0.05]) was statistically significantly increased in test group 2. Since this mid-dose value was within the historical control range (HCD: mean% 0.1 [0.0 – 2.0]) and the increase was not dose related, it was assessed as incidental.
The addition of skeletal malformations resulted in a slightly increased affected fetuses per litter incidence in test group 2 which attained statistical significance. However, due to the lack of dose-response relationship and the fact, that the overall incidences of all test groups were well within the historical control range (HCD total skeletal malformations: mean% 1.1 [0.0 – 3.9]), this increase was considered to be spontaneous in origin and not treatment-related.

For fetal skeletal variations and skeletal unclassified observations, see result chapter "Other effects".

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Weight of placentae:
The mean placental weight of test group 3 (1000 mg/kg bw/d) was statistically significantly lower in comparison to the concurrent control group. However, the high-dose mean value (0.37 g**, both sexes combined [p≤0.01]) was within the historical control range (HCD both sexes: mean 0.47 g [0.36 - 0.97]). Notably, for the dams, reduced food consumption and body weight gain was observed in the high dose group.
The mean placental weights of the low- and mid-dose groups (100 and 300 mg/kg bw/d) were comparable to the corresponding control group.

Fetal external variations:
No external variations were recorded.

Fetal external unclassified observations:
One external unclassified observation, placentae fused, was recorded in one mid-dose fetus without relation to dose (table 11). In addition, this finding can be found in the historical control data, thus, it was considered as not treatment-related.

Fetal soft tissue malformations:
Soft tissue malformations were detected in single fetuses of test groups 0, 2 and 3, as shown in tables 12 and 13. One high-dose fetus had an associated external malformation. These findings were single events in single fetuses and both can be found in the historical control data at comparable incidences. Thus, they were assessed as not treatment-related.
The total incidence of soft tissue malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data.

Fetal soft tissue variations:
Three soft tissue variations were detected: short innominate and dilated ureter in test groups 0 and 1, respectively, and dilated renal pelvis in test groups 0-2 (table 14). The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups. All of them can be found in the historical control data at comparable incidences. Therefore, they were assessed as not treatment-related.

Fetal soft tissue unclassified observations:
No soft tissue unclassified observations were recorded.

Fetal skeletal variations:
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dose (table 17). The overall incidences of skeletal variations were comparable to the historical control data. All skeletal variations with statistically significant differences between the control and any treated group were compiled in the table 18. All incidences were expressed on a fetus per litter basis and any statistically significant differences, which were outside the historical control range were marked in italicized bold types. As can be seen in table 18, the finding ‘incomplete ossification of sacral arch (cartilage present)’ was not related to dose and/or the mean values were clearly inside the historical control ranges. Therefore, this finding was assessed as not treatment-related.
The findings ‘unossified sternebra (unchanged cartilage)’ and ‘wavy rib’ were statistically significantly increased in test groups 2 and 3 (300 and 1000 mg/kg bw/d). The mid-dose values were clearly within the historical control range, respectively. All other skeletal variations recorded in the table 18 were statistically significantly increased in test group 3 (1000 mg/kg bw/d) and outside the respective historical control ranges.

Fetal skeletal unclassified cartilage observations:
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups (table 19). The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to the dose.

Details on embryotoxic / teratogenic effects:

Assessment of all fetal external, soft tissue and skeletal observations:
External, soft tissue and skeletal malformations were noted in one control, one low-dose, four mid-dose and three high-dose fetuses (0, 100, 300 and 1000 mg/kg bw/d). Four fetuses carried more than one malformation: for one male mid-dose fetus shortened scapula and bent rib was recorded, while one male mid-dose fetus showed a shortened scapula, bent rib and shortened humerus. One female high-dose fetus had anasarca combined with a situs inversus and for one male high-dose fetus a shortened scapula and a shortened humerus was recorded. The finding ‘bent rib’ which was seen in a multiple malformed fetus in test group 2 occurred also in two other fetuses of the same test group. Further malformations, i.e. hydronephrosis and misshapen basisphenoid, were observed in individual fetuses. These findings were single cases which occurred without relation to treatment and can be found in the historical control data. Since no ontogenetic pattern was recognizable in the other offspring of the test groups, these findings were regarded as not treatment-related. The total incidences of malformations are summarized in table 20.
External variations did not occur in any of the fetuses in this study. Three soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. None of the total incidences showed a relation to dosing (table 21). The majority of individual variations were equally distributed about the different test groups, if normal biological variation was taken into account, and can be found in the historical control data at a comparable frequency. The findings ‘unossified sternebra (unchanged cartilage)’ and ‘wavy rib’ were statistically significantly increased in test groups 2 and 3 (300 and 1000 mg/kg bw/d). The mid-dose values were clearly within the historical control range, respectively, and thus, evaluated as of no toxicological relevance. However, ‘incomplete ossification of supraoccipital (unchanged cartilage)’, ‘incomplete ossification of skull (unchanged cartilage)’, ‘incomplete ossification of nasal (unchanged cartilage)’, ‘basioccipital hole(s)’, ‘incomplete ossification of thoracic centrum (unchanged cartilage)’, ‘supernumerary thoracic vertebra’, ‘incomplete ossification of sacral arch (cartilage present)’, ‘unilateral ossification of sternebra (unchanged cartilage)’ and ‘incomplete ossification of pubis’ were statistically significantly increased in test group 3 (1000 mg/kg bw/d) and outside the respective historical control ranges. These findings indicated a slight delay in ossification, which is known to be reversible. Particularly, no changes in the underlying cartilages and no abnormalities in the respective bone structures were observed which qualifies them to be of relatively low toxicological concern. Ossification of skull bones and vertebral arches occurs at the end of gestation and the fetal ossification status is highly influenced by many factors, such as difference in mating time, time of cesarean section or by stress due to maternal toxicity (Carney & Kimmel, 2007). In this study, distinct maternal toxicity was observed at the highest-dose level. In addition, the observed skeletal variations did not influence the overall rate of fetal variations (table 21). Overall, the above-mentioned findings in the high-dose group (1000 mg/kg bw/d) were assessed as minor effects indicating a developmental delay and were, therefore, assessed as treatment-related but not as adverse. Soft tissue unclassified observations did not occur in any of the fetuses in this study. A spontaneous origin is assumed for the external unclassified observation and skeletal unclassified cartilage observations which were observed in several fetuses of all test groups (0, 100, 300 and 1000 mg/kg bw/d). The distribution and type of these findings did not suggest any relation to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

prenatal developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See "Remarks"

Abnormalities:

not specified

Developmental effects observed:

Table 7: Mean maternal body weights during gestation [in g]

			TEST GROUP 0 0 mg/kg bw/d		TEST GROUP 1 100 mg/kg bw/d	TEST GROUP 2 300 mg/kg bw/d	TEST GROUP 3 1000 mg/kg bw/d
DAY	0	MEAN	194.2	D	197.0	193.8	192.4
		S.D.	8.95		13.92	10.99	8.81
		N	23		25	25	23
DAY	1	MEAN	206.9	D	210.9	207.6	205.2
		S.D.	9.61		14.03	12.19	9.17
		N	23		25	25	23
DAY	3	MEAN	216.3	D	220.3	215.8	213.1
		S.D.	10.70		14.82	12.36	9.62
		N	23		25	25	23
DAY	6	MEAN	225.7	D	229.8	226.3	222.9
		S.D.	11.85		15.76	12.85	10.93
		N	23		25	25	23
DAY	8	MEAN	232.3	D	235.8	227.6	220.6*
		S.D.	12.09		16.68	13.66	9.37
		N	23		25	25	23
DAY	10	MEAN	240.3	D	244.1	235.8	221.0**
		S.D.	13.34		16.70	13.55	6.88
		N	23		25	25	23
DAY	13	MEAN	254.3	D	259.9	249.1	228.0**
		S.D.	14.38		18.32	14.71	9.84
		N	23		25	25	23
DAY	15	MEAN	265.1	D	271.4	260.5	237.5**
		S.D.	14.61		19.22	15.64	9.56
		N	23		25	25	23
DAY	17	MEAN	282.4	D	290.6	278.7	253.2**
		S.D.	16.68		19.87	16.96	10.82
		N	23		25	25	23
DAY	19	MEAN	305.4	D	314.7	302.5	274.4**
		S.D.	18.16		21.15	19.13	15.32
		N	23		25	25	23
DAY	20	MEAN	318.5	D	326.4	315.6	287.3**
		S.D.	18.37		22.54	21.01	17.52
		N	23		25	25	23

Statistics: D = Dunnett-test (two-sided); * : p <= 0.05, ** : p <= 0.01

Table 8: Mean maternal body weight change during gestation [in g]

					TEST GROUP 0 0 mg/kg bw/d		TEST GROUP 1 100 mg/kg bw/d	TEST GROUP 2 300 mg/kg bw/d	TEST GROUP 3 1000 mg/kg bw/d
DAYS	0	TO	1	MEAN	12.7	D	13.9	13.7	12.8
				S.D.	3.00		3.48	4.40	3.42
				N	23		25	25	23
DAYS	1	TO	3	MEAN	9.5	D	9.4	8.2	7.9
				S.D.	3.36		3.01	2.63	3.19
				N	23		25	25	23
DAYS	3	TO	6	MEAN	9.4	D	9.5	10.5	9.9
				S.D.	3.13		2.94	3.09	3.47
				N	23		25	25	23
DAYS	6	TO	8	MEAN	6.6	D	6.0	1.3**	-2.3**
				S.D.	2.28		3.23	3.74	5.78
				N	23		25	25	23
DAYS	8	TO	10	MEAN	8.0	D	8.3	8.2	0.3**
				S.D.	3.11		3.96	1.93	4.71
				N	23		25	25	23
DAYS	10	TO	13	MEAN	14.0	D	15.8	13.3	7.0**
				S.D.	3.18		3.28	3.76	8.25
				N	23		25	25	23
DAYS	13	TO	15	MEAN	10.8	D	11.6	11.4	9.5
				S.D.	2.11		2.60	3.67	5.67
				N	23		25	25	23
DAYS	15	TO	17	MEAN	17.3	D	19.2	18.2	15.7
				S.D.	3.77		4.45	3.53	5.94
				N	23		25	25	23
DAYS	17	TO	19	MEAN	22.9	D	24.1	23.8	21.2
				S.D.	3.98		4.90	4.54	8.32
				N	23		25	25	23
DAYS	19	TO	20	MEAN	13.2	D	11.7	13.1	12.9
				S.D.	2.87		4.07	4.80	4.81
				N	23		25	25	23

Statistics: D = Dunnett-test (two-sided); * : p <= 0.05, ** : p <= 0.01

Table 9: Individual fetal external malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	79-06 Fa)	Anasarca

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a) fetus with additional soft tissue malformation

Table 10: Total external malformations

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter

Fetuses

259

278

290

259

Fetal incidence

N (%)

0.0

1 (0.4)

Litter incidence

N (%)

0.0

1 (4.5)

Affected fetuses/litter

Mean%

0.0

0.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 11: Total external unclassified observations

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter

Fetuses

259

278

290

259

Fetal incidence

N (%)

0.0

1 (0.3)

0.0

Litter incidence

N (%)

0.0

1 (4.0)

0.0

Affected fetuses/litter

Mean%

0.0

0.3

0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 12: Individual fetal soft tissue malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	6-04 M	Hydronephrosis
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	53-14 M	Situs inversus
3 (1000 mg/kg bw/d)	79-06 Fa)	Situs inversus

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a) fetus with additional external malformation

Table 13: Total soft tissue malformations

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter

Fetuses

124

132

139

122

Fetal incidence

N (%)

1 (0.8)

0.0

1 (0.7)

1 (0.8)

Litter incidence

N (%)

1 (4.3)

0.0

1 (4.0)

1 (4.5)

Affected fetuses/litter

Mean%

0.6

0.0

0.5

0.8

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 14: Total soft tissue variations

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter

Fetuses

124

132

139

122

Fetal incidence

N (%)

5 (4.0)

6 (4.5)

1 (0.7)

0.0

Litter incidence

N (%)

3 (13)

5 (20)

1 (4.0)

0.0

Affected fetuses/litter

Mean%

3.8

4.0

0.8

0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 15: Individual fetal skeletal malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	34-11 M	Misshapen basisphenoid
2 (300 mg/kg bw/d)	58-11 M	Shortened scapula, bent rib
	59-01 M	Shortened scapula, bent rib, shortened humerus
	62-01 F	Bent rib
3 (1000 mg/kg bw/d)	83-01 M	Shortened scapula, shortened humerus
	88-13 M	Shortened scapula

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Table 16: Total skeletal malformations

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter

Fetuses

135

146

151

137

Fetal incidence

N (%)

0.0

1 (0.7)

3 (2.0)

2 (1.5)

Litter incidence

N (%)

0.0

1 (4.0)

3 (12)

2 (9.1)

Affected fetuses/litter

Mean%

0.0

0.7

2.0*

1.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

* = p <= 0.05 (Wilcoxon-test [one-sided])

Table 17: Total fetal skeletal variations

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter

Fetuses

135

146

151

137

Fetal incidence

N (%)

134 (99)

142 (97)

146 (97)

137 (100)

Litter incidence

N (%)

23 (100)

25 (100)

22 (100)

Affected fetuses/litter

Mean%

99.4

97.3

96.8

100.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 18: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

HCD

Mean % (range)

Incomplete ossification of supraoccipital; unchanged cartilage

22.7

19.5

27.7

56.4**

21.6

(9.1 - 32.4)

Incomplete ossification of skull; unchanged cartilage

12.2

13.3

16.2

26.3*

10.1

(2.1 - 21.5)

Incomplete ossification of nasal; unchanged cartilage

0.7

0.6

2.7

11.7**

0.9

(0.0 - 4.1)

Basioccipital hole(s)

0.0

2.4*

0.4

(0.0 - 1.5)

Incomplete ossification of thoracic centrum; unchanged cartilage

0.7

0.0

7.0*

0.4

(0.0 - 2.0)

Supernumerary thoracic vertebra

1.5

9.9*

3.4

12.7**

5.6

(3.0 - 10.3)

Incomplete ossification of sacral arch;

cartilage present

0.0

2.6*

1.6

0.8

(0.0 - 2.7)

Unossified sternebra; unchanged cartilage

2.7

5.8

9.0*

26.0**

5.2

(0.0 - 13.8)

Unilateral ossification of sternebra;

unchanged cartilage

2.1

0.0

2.7

8.1*

0.6

(0.0 - 2.2)

Wavy rib

0.9

9.9

10.9**

18.2**

4.7

(0.7 - 14.7)

Incomplete ossification of pubis; cartilage present

0.0

0.6

0.0

3.7*

0.3

(0.0 - 1.0)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p <= 0.05 (Wilcoxon-test [one-sided]) ** = p <= 0.01 (Wilcoxon-test [one-sided])

Table 19: Total unclassified cartilage observations

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter

Fetuses

135

146

151

137

Fetal incidence

N (%)

105 (78)

116 (79)

98 (65)

100 (73)

Litter incidence

N (%)

23 (100)

25 (100)

22 (100)

Affected fetuses/litter

Mean%

77.6

81.0

65.1

73.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 20: Total fetal malformations

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter

Fetuses

259

278

290

259

Fetal incidence

N (%)

1 (0.4)

4 (1.4)

3 (1.2)

Litter incidence

N (%)

1 (4.3)

1 (4.0)

4 (16)

3 (14)

Affected fetuses/litter

Mean%

0.3

0.4

1.3

1.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 21: Total fetal variations

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter

Fetuses

259

278

290

259

Fetal incidence

N (%)

139 (54)

148 (53)

147 (51)

137 (53)

Litter incidence

N (%)

23 (100)

25 (100)

22 (100)

Affected fetuses/litter

Mean%

53.6

53.3

50.8

52.9

mg/kg bw/d = milligram per kilogram body weight per day; N= number; % = per cent

Conclusions:

The no observed adverse effect level (NOAEL) for maternal toxicity is the mid-dose level of 300 mg/kg bw/d. The NOAEL for prenatal developmental toxicity is 1000 mg/kg bw/d. The test substance is considered not teratogenic in Wistar rats under the test conditions employed in this study.

Executive summary:

In this GLP-compliant prenatal developmental toxicity study, the test was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity.

Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

Regarding clinical examinations, all females of the test substance treated groups (test group 1-3; 100, 300 or 1000 mg/kg bw/d) showed yellowish discolored feces during the treatment period until terminal sacrifice (GD 20). It was initially observed on GD 7 in the mid- and high-dose groups and on GD 8 in the low-dose group. This feces discoloration mirrored the presence of the yellowish test substance in the gastrointestinal tract. An adverse, toxic effect by itself was not assumed.

At the high-dose level (1000 mg/kg bw/d), distinct signs of maternal toxicity were observed such as piloerection from GD 10 onwards in all dams and hypothermia on GD 10-12 in one dam. In addition, reduced mean food consumption (29% below control) and decreased body weight change (35% below control) were seen during the treatment period (GD 6-19). The corrected body weight gain of these high-dose dams was also statistically significantly decreased (69% below control). Due to the variety of affected parameters, the abovementioned findings were assessed as treatment-related and adverse.

Although the dams of the test group 2 (300 mg/kg bw/d) showed a decreased mean food consumption on GD 6-13 (up to 16% below control), they recovered afterwards and even exceeded the control value at the end of the treatment period. Piloerection was seen on GD 19-20 in one dam only. These findings were considered as signs of a beginning maternal toxicity, which were related to the treatment but not yet adverse.

All in all, at 300 and 100 mg/kg bw/d, no treatment-related adverse effects were observed. Regarding clinical pathology, in dams of test group 3 (1000 mg/kg bw/d) significantly increased total bilirubin levels and significantly decreased total protein, albumin and globulin levels, in absence of any signs of anemia, were observed most probably due to an altered liver cell metabolism. These findings were regarded to be treatment-related and adverse.

Regarding pathology, all findings were considered to be incidental or spontaneous in origin and without any relation to treatment.

No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution was noted. However, anogenital distance and anogenital index were decreased in male fetuses (-19% and -14% below control, respectively) of the high-dose group. In addition, lower mean placental weights (0.37 g vs. 0.45 g on control, both sexes combined) and mean fetal body weights (3.1 g vs. 3.6 g in control, all viable fetuses) were observed in this group (1000 mg/kg bw/d). It was assumed that these parameters were possibly affected by treatment, but since almost all of them were within the historical control data regarded to be not adverse.

The observed reduced fetal body weights of the high-dose group (1000 mg/kg bw/d) corresponded to a delay in ossification as indicated by a broad range of skeletal variations, i.e. ‘unossified sternebra (unchanged cartilage)’, ‘wavy rib’, ‘incomplete ossification of supraoccipital (unchanged cartilage)’, ‘incomplete ossification of skull (unchanged cartilage)’, ‘incomplete ossification of nasal (unchanged cartilage)’, ‘basioccipital hole(s)’, ‘incomplete ossification of thoracic centrum (unchanged cartilage)’, ‘supernumerary thoracic vertebra’, ‘incomplete ossification of sacral arch (cartilage present)’, ‘unilateral ossification of sternebra (unchanged cartilage)’ and ‘incomplete ossification of pubis’. Almost all of these variations appeared at high background incidences in the population of the used rat breed, are unlikely to be detrimental for survival or health and are at least to some extent being repaired postnatally, which qualifies them to be of relatively low toxicological concern. Specifically, the ossification of skull bones and vertebral arches occurs at the end of gestation and the fetal ossification status is highly influenced by many factors, such as difference in mating time, time of cesarean section or by stress due to maternal toxicity (Carney & Kimmel, 2007). In this study, the findings in the high-dose fetuses occurred in presence of maternal toxicity and were in the same order of magnitude as reduced food consumption and body weight gain of the dams. Overall, the above-mentioned findings were considered to be minor, reversible effects indicating a developmental delay and were, therefore, assessed as treatment-related but not as adverse.

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) provided evidence of maternal toxicity, such as a reduction in food consumption, a decrease in (corrected) body weight gain and changes in clinical pathology parameters (e.g. increased total bilirubin values) at the highest dose level of 1000 mg/kg bw/d. In conclusion, the NOAEL for maternal toxicity is the mid-dose level of 300 mg/kg bw/d.

Overall, there was no evidence for specific adverse effects of the test substance on fetal morphology at any dose. Minor, reversible effects on fetal weight and ossification in the presence of maternal toxicity were indicating a temporary developmental delay and were, therefore, assessed as treatment-related but not as adverse. Thus, the NOAEL for prenatal developmental toxicity is 1000 mg/kg bw/d. The test substance is considered not teratogenic in Wistar rats under the test conditions employed in this study.

Reference 2

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:

Specific details on test material used for the study:

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% sodium carboxymethyl cellulose suspension in deionized water

Details on exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Details on mating procedure:

Duration of treatment / exposure:

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:

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):

Other effects:

no effects observed

Description (incidence and severity):

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):

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)

(100)

(300)

(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:

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.

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

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Species:: rat

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

Key study: Developmental toxicity study

Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

Regarding pathology, all findings were considered to be incidental or spontaneous in origin and without any relation to treatment.

Justification for classification or non-classification

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

The available screening study is reliable and suitable for classification purposes under Regulation 1272/2008. No adverse effects on fertility were observed in a screening study in rats (OECD 421) and no adverse effects were observed in the fetuses in a teratogenicity/developmental toxicity study in rats (OECD 414). As a result, the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the seventeenth time in Regulation (EC) No. 2021/849.

During the thirteen days covered in the screening study, no effects via lactation were observed.

Additional information

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Registration Dossier

Registration Dossier

Data platform availability banner - registered substances factsheets

Diss Factsheets

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

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Reference

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Justification for classification or non-classification

Additional information

Referenceopen all close all