4,11-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD TG 421, GLP, rat: NOAEL = 1000 mg/kg bw

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep 20, 2021 - Nov 04, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL
- Batch number: 200389
- Purity: 94.11 area-% (HPLC, 300 nm), 94.82 area-% (HPLC, 335 nm)
- Physical state/ appearance: solid/ red

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition: room temperature
- Stability: expiry date: 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
- Homogeneity: given
- Stability in the medium: the stability of the test substance in 0.5 % Sodium carboxymethyl cellulose in deionized water was demonstrated for a period of 7 days at room temperature
- Solubility and stability of the test material in the solvent/vehicle: the test substance reveals the characteristics of a pigment which is not soluble in water

FORM AS APPLIED IN THE TEST
- As a suspension

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, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 13 - 15 weeks
- Weight at study initiation: weight variation did not exceed 20 % of the mean weight of each sex
- Housing: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm² (610 x 435 x 215 mm); supplied by TECHNIPLAST (Hohenpeißenberg, Germany) during pretreatment (up to 5 animals per sex and cage), during premating (2 animals per sex and cage), during mating and postmating (2 animals per cage (males only))
- Diet: mouse and rat maintenance diet “GLP”, meal, supplied by Granovit AG (Kaiseraugst, Switzerland), ad libitum
- Water: drinking water (from water bottles), ad libitum
- Acclimation period: about 3 weeks

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: Oct 12, 2021 To: Dec 09, 2021

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test substance suspensions in 0.5 % carboxymethyl cellulose (CMC) in deionized water were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a graduated beaker depending on the dose group, topped up with 0.5% CMC in deionized water and intensely mixed with a homogenizer until it was homogeneous visually. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0
- Paired for a maximum of 2 weeks
- After successful mating each pregnant female was caged: Polycarbonate cages type III females during mating, gestation, lactation and after weaning (1 animal per cage with following xceptions: during overnight mating:1 male/1 female per cage and during rearing up to PND 13: 1 dam with her litter)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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. Of each sample, one additional reserve sample (described by the suffix “R”) was retained. The samples collected at the beginning and during lactation period were analyzed in the Analytical Laboratory. Due to equivocal results additionally, the reserve samples of the highest concentration and two reserve samples of the lowest concentration (top and bottom) collected at the start of the study, all samples and one reserve sample of the highest concentration (bottom) collected at the beginning of gestation and reserve samples of the lowest (top and bottom), mid and highest concentration (bottom) collected towards the end of the administration period were analyzed. All further samples were not analyzed and stored frozen (at -20°C) in the Laboratory of the Mechanistic Toxicology until the finalization of the study. Following finalization of the report, all analytical samples, including reserve samples, will be discarded.
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 widening of the target range to 80 - 120 % can be considered acceptable. Most measured values were in the expected range of 80 - 120 %, demonstrating the correctness of the preparations. However, deviations from these target values occurred: Values of the measured samples collected at the start of the administration period varied from 50 - 90 %. Therefore some of the reserve samples were analyzed. The values of the reserve samples were found in the range of 86 - 101 % with one exception (sample No. 4R: 65 %). The values of the samples collected at the beginning of gestation were found in the range of
91 - 100 %. (Note: Sample No. 16 revealed 8 0%, for the reserve sample 16R a value of 100 % was found). The values of the measured samples collected towards the end of the administration period varied from 61 - 101 %. Therefore some of the reserve samples were analyzed. The values
of the reserve samples were found in the range of 84 - 97 %.

Duration of treatment / exposure:

The duration of treatment covered 30 days in-life period (males) including 4 days mating (mating pairs were from the same test group) as well as a 2-weeks premating period (females), 4 days mating period, the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.

Frequency of treatment:

dailey, 7 days each week

Details on study schedule:

- The females were allowed to deliver and rear their pups until PND 4 (standardization) or PND 13

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: at the request of the sponsor, the following dose levels were selected
- Fasting period before blood sampling for clinical biochemistry: 16 -20 h

Positive control:

no

Parental animals: Observations and examinations:

MORTALITY
- Time schedule: twice daily on working days or once daily (Saturday, Sunday or on public holidays)

CLINICAL OBSERVATIONS
- Time schedule: 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, 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

BODY WEIGHT
- Time schedule: weekly

FOOD CONSUMPTION
- Time schedule: weekly

WATER CONSUMPTION
- Time schedule: daily
- Visual inspection of the water bottles for any changes in volume.

THYROID HORMONES
- Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia
- The animals were fastened before blood sampling
- Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4 and TSH)
- All generated serum samples were frozen at -80°C until measurement
- The assays of serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results
- The results of clinical pathology examinations were expressed in International System (SI) units
- The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany)
- T4 Elisa was measured with a Sunrise MTP-reader (Tecan AG, Maennedorf, Switzerland), and evaluated with the Magellan-Software of the instrument producer
- 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 will be frozen at -80°

Oestrous cyclicity (parental animals):

For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization. For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Sperm parameters (parental animals):

not examined

Litter observations:

STANDARDISATION OF LITTERS
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Surplus pups were sacrificed. Standardization of litters was not performed in litters with ≤ 8 pups.

PUP NUMBER AND STATUS AT DELIVERY
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

SEX RATIO
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 13 after birth according to the following formula:

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

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

PUP BODY WEIGHT DATA
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters.“Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75 % of the mean weight of the respective control pups.

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

ANOGENITAL INDEX
The anogenital index was calculated according to the following formula:

anogenital index = anogenital distance [mm] / cubic root of pup weight [g]

NIPPLE/AREOLA
All surviving male pups were examined for the presence of nipples/areolas on PND 13. The number of nipple/areola was counted.

THYROID HORMONES
- Blood samples were taken from all surplus pups per litter at PND 4 by decapitation under isoflurane anesthesia
- Blood samples from the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH)
- All generated serum samples were frozen at -80°C until measurement
- The assays of serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results
- The results of clinical pathology examinations were expressed in International System (SI) units
- The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany)
- T4 Elisa was measured with a Sunrise MTP-reader (Tecan AG, Maennedorf, Switzerland), and evaluated with the Magellan-Software of the instrument producer
- In the samples of pups at PND 4 no thyroid hormones were measured, because no relevant changes of T4 and TSH values in pups at PND13 were observed
- For the same reason, no T3 values were measured in any sample collected for thyroid hormone measurement
- All generated serum samples will be frozen at -80° at least

Postmortem examinations (parental animals):

NECROPSY
The male and female animals were sacrificed 30 and 58 days, respectively, after the beginning
of the administration, and examined. All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

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
The uteri of all cohabited female parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1 % ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.

HISTOPATHOLOGY
Fixation was followed by histotechnical processing, examination by light microscopy and assessment
of findings in testes, epididymides and ovaries. The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004). Special attention was given to the stages of spermatogenesis and histopathology of interstitial testicular cell structure. Whenever the diagnosis „no abnormalities detected” was used in the ovary, it implies that all the different stages of functional bodies (especially corpora lutea) were present and normal. A correlation between gross lesions and histopathological findings was attempted.

Postmortem examinations (offspring):

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.

Statistics:

Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights,the litter means were used), gestation days, anogenital distance, anogenital index:
Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means

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

Mating days until day 0 pc, % postimplantation loss, pups stillborn, % perinatal loss, nipple development, Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival index:
Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians

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

Number of cycles and cycle length, blood samples with bidirectional changes, organ weights:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians

Blood samples with unidirectional changes:
Pair-wise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Terminal body weight:
Comparison of each group with the control group was performed using the DUNNETT-test (two-sided) for the hypothesis of equal means

Reproductive indices:

MALE
The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs. For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:

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


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
The pairing partners, the number of mating days until vaginal sperm was detected and gestational status was recorded for F0 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

The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:

Live birth index (%) = number of liveborn pups at birth / total number of pups born x 100

The implantations were counted and the postimplantation loss (in %) was calculated according
the following formula:

Postimplantation loss (%) = number of implantations – number of pups delivered / number of implantations x 100

Offspring viability indices:

In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in section “Post mortem examinations (offspring)”. 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)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test substance-related, adverse findings were observed in any test group. Male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) showed red discolored feces (test substance-like). Discolored feces were observed in male and female animals of mid-dose and high-dose groups from study day 1 onwards, in low-dose males on the last two study days (day 29 - 30) in low-dose females from GD 13 - 15 onwards. The finding was clearly related to the test substance but assessed as being not adverse. Mid-dose male animal No. 21 showed respiration sounds on study day 12, only. This finding was considered as caused by treatment but not related to the test substance. Delivery was altered in individual female animals, but a relation to dosing was not considered: Female animal No. 110 of the control group showed pale skin on PND 0, about 50 % of the pups were stillborn. It was concluded that this individual had problems to deliver. For female animal No. 128 of the mid-dose group, blood in bedding was recorded on GD 23 to 24 and a mass was palpable in abdomen on the same days. On GD 24, the animal showed bloody vaginal discharge. On GD 25, the palpable mass had disappeared. No pups were found but the animal had implants. For female animal No. 133 of the high dose group, blood in bedding, piloerection and slightly labored respiration was recorded on GD 23. On the next day (PND 0) all pups of this dam were found stillborn and the female showed pale skin from PND 0 to 1. These findings were considered as spontaneous in nature and not treatment-related. As an animal of the control group showed the same pattern of findings it was concluded that dystocia had a genetic background in this batch of animals.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

THYROID HORMONES
In parental male (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.

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: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

For all F0 parental males of test groups 0 to 2, which were placed with females to generate F1 pups, copulation was confirmed. In test group 3, copulation could not be confirmed for male animal No. 32. Thus, the male mating index was 100 % in test groups 0 to 2 and 90 % in test group 3. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter, only high-dose male No. 32 did not generate pups. Thus, the male fertility index was 100 % for test groups 0 to 2 and 90 % in test group 3. Both indices reflect the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rats did not show relevant gross lesions The female mating index calculated after the mating period for F1 litter was 100 % in test groups 0 to 2 and 90 % in test group 3. The mean duration until sperm was detected (GD 0) varied between 1.6 and 2.1 days what reflected the normal range of biological variation inherent in the strain of rats used for this study. All female rats delivered pups or had implants in the utero with the exception of female animal No. 132 (mated with male animal No. 32) which did not become pregnant. The fertility index was 100 % for test groups 0 to 2 and 90 % in test group 3. The non-pregnant female did not show any relevant gross or histopathological findings. The mean duration of gestation values varied between 22.0 (control), 22.1 (test group 1), 22.1 (test group 2) and 22.4 (test group 3). The gestation index was 100 % in control and test group 1, 90 % in test group 2 and 77.8 % in test group 3. The number of females with stillborn pups was comparable in all test groups and reflected the normal range of biological variation inherent in the strain of rats used for this study. Implantation was not affected by 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 (14.7 / 14.1 / 11.6 and 12.2 implants/dam in test groups 0, 1, 2 and 3, respectively). In test group 1, postimplantation loss was comparable to control (2.5 % vs. 4.6%). Postimplantation loss was increased in test groups 2 and 3 (15.2 and 12.6 %) compared to control (4.6 %) but these values were still within the range of historical control data (0.9 - 16.8 %). It was considered that there was no test substance-induced intrauterine embryo-/fetolethality. The rate of liveborn pups for test groups 1 and 2 were 99.3 and 99.1 %. In test group 3, the rate was reduced (88.7 %) but comparable to control group (92.9 %). The number of stillborn pups and mean was 10 / 1 / 1 / 12 and 1.0 / 0.1 / 0.1 / 1.5 in test groups 0, 1, 2, and 3, respectively. In test group 3, there was one female (No. 133) with stillborn pups, only. However, values in test group 3 were only slightly higher than in control group and in historical control data (mean of means: 1.3). The perinatal loss was 5.5 % / 0.8 % / 2.8 % and 14.2 % in test groups 0 to 3, respectively. Perinatal loss in test group 3 was increased and slightly higher than the range of historical control data (0.0 - 12.8 %). As an animal of the control group showed the same pattern of findings, i.e., labored birth, it was concluded that dystocia had a genetic background in this batch of animals. Thus, the test substance did not adversely affect reproduction and delivery of the F0 generation parental females.

Regarding clinical examinations, signs of general systemic toxicity were not observed in male
or female parental animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) during the entire
study period. Reddish discolored feces occurred in all test substance-treated test groups and were assessed to be related to the test group but non-adverse in its nature. Delivery was altered in individual female animals, but a relation to dosing was not considered because female animal No. 110 of the control group seemed to have problems to deliver as it was observed for female animal No. 128 of the mid-dose group, and female animal No. 133 of the high-dose group. As the mentioned animal of the control group showed signs of dystocia, i.e., pale skin and stillborn pups, a genetic background in this batch of animals was considered to be the cause. Fertility indices for male and female animals were not impaired by test-substance administration even at a dose level of 1000 mg/kg bw/d. In addition, live birth indices of pups in all test groups were not influenced by the test substance. 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, target organ was the gastrointestinal tract with several compartments showing discoloured content in test groups 2 and 3. This was considered treatment-related, but not adverse as clinically no functional impairment of the gastrointestinal tract (e.g., weight loss) was observed. 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.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects observed up to the highest tested dose

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects observed up to the highest tested dose

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related, adverse effects on pup body weight development were observed in any test group. In test group 2 (300 mg/kg bw/d), mean pup body weights were significantly higher compared to the concurrent control values with regard to female pups on PND 7 and 13 (+5.7 and +7.4 %, respectively), male pups on PND 13 (5 %) and both sexes combined on PND 7 and 13 (+5.4 and +6.8 %, respectively). The mean pup body weights in the low- and high-dose groups as well as pup body weight change values in all dose groups of male and female pups and both sexes combined were comparable to the concurrent control values.
Two male runts were observed in test group 1 and two female runts were observed in test group 3.

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:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related, adverse findings were observed in pups of any test group. A few pups showed findings at gross necropsy, such as discolored liver, empty stomach, iscolored
testis, limb lesion, dilated renal pelvis and post-mortem utolysis or were partly cannibalized. 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:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

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

The viability index as indicator for pup mortality was not altered.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects observed up to the highest tested dose

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

NOAEL (systemic) = 1000 mg/kg bw, NOAEL(reproductive) = 1000 mg/kg bw, NOAEL (developmental) = 1000 mg/kg bw

Executive summary:

In a  reliable GLP-conform study according to OECD TG 421, the test substance was examined for its possible effects on the integrity and performance of the male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition. The test substance was administered daily by gavage as a suspension in deionized water containing 0.5% Sodium carboxymethyl cellulose to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0, 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (deionized water containing 0.5 % Sodium carboxymethyl cellulose). The duration of treatment covered 30 days in-life period (males) including 4 days mating (mating pairs were from the same test group) as well as a 2-weeks premating period (females), 4 days mating period, the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. Food consumption of the F0 parents was determined once weekly during premating. In dams, food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4, 4 - 7, 7 - 10 and 10 - 13. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the day of parturition (postnatal day [PND] 0) and on PND 1, 4, 7, 10 and 13. Estrous cycle data were evaluated for F0 generation females over a two-weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4, 7 and 13. Their viability was recorded. At necropsy on PND 13, all pups were sacrificed under isoflurane anesthesia with CO2 (except the selected pups for blood sampling) and examined macroscopically for external and visceral findings. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted. Blood samples were taken from all surplus pups at PND 4 as well as from one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. All F0 parental  nimals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded, and histopathological examination was performed.

 

The various analyses confirmed the stability of the test substance preparations over a period of 7 days at room temperature and the correct concentrations in the test substance preparations taking into account that the test substance is characterized as insoluble pigment. The following test substance-related, relevant effects/findings were noted:

 

Test group 3: 1000 mg/kg bw/d

F0 PARENTAL ANIMALS

• No test substance-related, adverse findings were observed.

F1 PUPS

• No test substance-related, adverse findings were observed.

 

Test group 2: 300 mg/kg bw/d

F0 PARENTAL ANIMALS

• No test substance-related, adverse findings were observed.

F1 PUPS

• No test substance-related, adverse findings were observed.

 

Test group 1: 100 mg/kg bw/d

F0 PARENTAL ANIMALS

• No test substance-related, adverse findings were observed.

F1 PUPS

• No test substance-related, adverse findings were observed.

 

Under the conditions of the present Reproduction/Developmental Toxicity Screening Test the oral administration of the test substance to male and female Wistar rats did not induce signs of systemic toxicity even at the highest dose level tested, i.e. 1000 mg/kg bw/d. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats. The no observed adverse effect level (NOAEL) for fertility and reproductive performance was 1000 mg/kg bw/d for male and female Wistar rats. The no observed adverse effect level (NOAEL) for developmental toxicity was 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

Quality of whole database:

OECD TG 421, GLP, K1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a  reliable GLP-conform study according to OECD TG 421, the test substance was examined for its possible effects on the integrity and performance of the male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition. The test substance was administered daily by gavage as a suspension in deionized water containing 0.5% Sodium carboxymethyl cellulose to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0, 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (deionized water containing 0.5 % Sodium carboxymethyl cellulose). The duration of treatment covered 30 days in-life period (males) including 4 days mating (mating pairs were from the same test group) as well as a 2-weeks premating period (females), 4 days mating period, the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. Food consumption of the F0 parents was determined once weekly during premating. In dams, food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4, 4 - 7, 7 - 10 and 10 - 13. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the day of parturition (postnatal day [PND] 0) and on PND 1, 4, 7, 10 and 13. Estrous cycle data were evaluated for F0 generation females over a two-weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4, 7 and 13. Their viability was recorded. At necropsy on PND 13, all pups were sacrificed under isoflurane anesthesia with CO2 (except the selected pups for blood sampling) and examined macroscopically for external and visceral findings. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted. Blood samples were taken from all surplus pups at PND 4 as well as from one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. All F0 parental  nimals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded, and histopathological examination was performed.

 

The various analyses confirmed the stability of the test substance preparations over a period of 7 days at room temperature and the correct concentrations in the test substance preparations taking into account that the test substance is characterized as insoluble pigment. The following test substance-related, relevant effects/findings were noted:

 

Test group 3: 1000 mg/kg bw/d

F0 PARENTAL ANIMALS

• No test substance-related, adverse findings were observed.

F1 PUPS

• No test substance-related, adverse findings were observed.

 

Test group 2: 300 mg/kg bw/d

F0 PARENTAL ANIMALS

• No test substance-related, adverse findings were observed.

F1 PUPS

• No test substance-related, adverse findings were observed.

 

Test group 1: 100 mg/kg bw/d

F0 PARENTAL ANIMALS

• No test substance-related, adverse findings were observed.

F1 PUPS

• No test substance-related, adverse findings were observed.

 

Under the conditions of the present Reproduction/Developmental Toxicity Screening Test the oral administration of the test substance to male and female Wistar rats did not induce signs of systemic toxicity even at the highest dose level tested, i.e. 1000 mg/kg bw/d. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats. The no observed adverse effect level (NOAEL) for fertility and reproductive performance was 1000 mg/kg bw/d for male and female Wistar rats. The no observed adverse effect level (NOAEL) for developmental toxicity was 1000 mg/kg bw/d.

Effects on developmental toxicity

Description of key information

No study available, but in a screening test according to OECD TG 421 no developmental effects in the offspring could be observed up to the highest test dose (1000 mg/kg bw).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

only screening study available (OECD TG 421, GLP, K1)

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

4, 11 -Dichloroquinacridone has not been tested for its reproductive toxicity – developmental toxicity. However, in a screening test according to OECD TG 421 no developmental effects could be observed up to the highest test dose (1000 mg/kg bw) in the offspring.

 

Additionally, there are several reliable data available for other quinacridone pigments (Pigment Violet 19 and Pigments Red 122).

In a reliable GLP-conform study according to OECD TG 414 (Klimisch score 1), Pigment Violet 19 diluted in 0.5% CMC was administered by gavage to female Sprague Dawley rats at doses of 111, 333 and 1000 mg/kg bw once daily from gestation day 5 to 19. No toxicologically relevant changes were observed and a NOAEL of 1000 mg/kg bw can be concluded.

In a reliable GLP-conform study according to OECD TG 414 (Klimisch score 1), Pigment Red 122 diluted in 0.5% CMC was administered by gavage to female Sprague Dawley rats at doses of 111, 333 and 1000 mg/kg bw once daily from gestation day 5 to 19. No toxicologically relevant changes were observed and a NOAEL of 1000 mg/kg bw can be concluded.

Therefore, no classification for reproduction toxicity – developmental toxicity is necessary for the members of the Quinacridone Pigments category.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not classified for toxicity to reproduction under Regulation (EC) No. 1272/2008, as amended for the 17th time in Regulation (EC) No. 2021/849.