N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The OECD 421 was not part of the requests concerning the Final Decision of a Compliance Check CCH-D-2114476180-53-01/F. However, it was performed to provide sufficient information on development and reproduction in order to strengthen a read-across approach to an analogue substance. If the data would not support the read-across justification, the registrant would perform the OECD 443. This was decided during a phone call with ECHA representatives.

The results of a reproduction/developmental toxicity screening study (OECD 421) with the test item in rats by the oral route indicate that a potential to impair fertility cannot be fully excluded for high doses. The NOAEL for adult fertility and reproduction parameters as well as for pups was set to 225mg/kg bw/day for males and females. The obtained data from the OECD 421 study did not strengthen the provided read-across justification. As the significance of the findings for human health is not clear at this time, an Extended-One-Generation study (OECD 443) was initiated and the final study report is planned for 15^th August 2023 according to the study protocol. An update of all relevant information will be submitted at the earliest date possible but not earlier than Q3 2023.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Batch number: Batch 119 Tin-free
- Expiry date: 30 November 2023
- Purity: 89.3 area % (HPLC, 254 nm), 94.3 area % (HPLC, 230 nm)
- Appearance: Transparent to opaque, slightly yellow, apparently homogeneous solid (granular-like)
- Storage conditions: room temperature

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and because there are ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Calco, Italy (breeder: Charles River Laboratories, Domaine des Oncins, 69210 Saint Germain Nuelles, France)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: Males: 209.8-222.5 g; Females: 172.0-201.3 g
- Housing:
From arriving to mating: up to 5 animals of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags.
During mating: one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada
S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material.
After mating: males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags.
- Diet: ad libitum (laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy))
- Water: ad libitum (drinking water)
- Acclimation period: 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2
- Humidity (%): 55 ±15
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 02 September 2020 (=Date of dosing - acclimatisation period) to 21 Dezember 2020 (Dams and litter necropsy)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in the vehicle according to the following procedure:
- the required amount of powder was weighed;
- the powder was transferred to a mortar and, with the aid of pestle, the granularity of the test item was reduced to a small size;
- the test item was transferred to a glass container;
- the vehicle was added very slowly;
- the obtained suspensions were then homogenised (by Silverson) for approximately 5 minutes and left overnight on a magnetic stirrer at room temperature prior to use and up until the time of dosing of the last animal.

The preparations were made weekly or daily (concentrations of 7.5, 22.5 and 75.0 mg/mL), according to stability data and phase of the study.


VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until positive identification occurred or for a maximum of 2 weeks
- Proof of pregnancy: sperm, vaginal plag in situ or copulation plugs were used as positive identification of copulation. Vaginal plug in situ is considered as "1" for calculation.
- After successful mating each pregnant female was caged (how): They were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags.

- Any other notifications: Due to premature death of one male during premating phase, one male which had already mated was paired (1:1) with a second female, such that all females were paired.
Further, one female and one male were separate after 14 days of cohabitation due to failed mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A 28 hour stability at room temperature and an 8 day stability at +5°C ± 3°C were verified in the range from 0.2 to 200 mg/mL.

The preparations were made weekly or daily (concentrations of 7.5, 22.5 and 75.0 mg/mL).
The proposed preparation procedure for the test item was checked for preparations at 0.2 and 200 mg/mL by chemical analysis (concentration and homogeneity). Final results for all levels were within the acceptability limits for concentration (85-115%) and homogeneity (CV < 10%).

Duration of treatment / exposure:

- males: 30 days. The duration of treatment covered a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.
- females: 51, 55 and 57 days. The duration of treatment covered a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice.

Frequency of treatment:

daily

Dose / conc.:

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

Remarks:

Test group 1 (Control)

Dose / conc.:

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

Remarks:

Test group 2

Dose / conc.:

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

Remarks:

Test group 3

Dose / conc.:

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

Remarks:

Test group 4

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on information from a preliminary, non GLP compliant study (ERBC Study Number Y0520). In this study, signs of toxicity were noted at the dose level of 1000 mg/kg/day. Decreased activity and slight to moderate piloerection were noted in females, starting from Day 6 of the gestation period. On the basis of these results, 750 mg/kg/day was considered a suitable high dose level in this study.
- Fasting period before blood sampling for clinical biochemistry: not specified

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Mortality: Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day.
- Clinical observations: Observations were performed at the same time interval each day, the interval was selected and changed taking into consideration the presence of post-dose reactions. Clinical observations were done as follows: > approximately 30 minutes - 1 hour post-dose from the beginning of the study up to Day 7 of the mating period; > within 30 minutes post-dose from Day 8 of the mating period up to the end of the study.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: once a week
- Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum.
- Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.

FOOD CONSUMPTION: Yes
- Time schedule: weekly during the pre-mating period, starting from allocation.
- Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum, and on Days 7 and 13 post partum, starting from Day 1 post partum.

WATER CONSUMPTION (if drinking water study): No

CLINICAL BIOCHEMISTRY: Yes
- Anaesthetic used for blood collection (for hormone determination) : Yes (Isoflurane)
- How many animals: all parental males and females
- Parameter checked: T4, TSH (serum)


REPRODUCTIVE PERFORMANCE
- Mating: 1:1
- Parturition and gestation length: A parturition check was performed from Day 20 to Day 25 post coitum. Three females which did not gave birth after 25 days of post coitum period were sacrificed shortly after and the uteri were immersed in a 20% solution of ammonium sulphide to reveal evidence of early stages of implantation. One female that did not mate was sacrificed 30 days after the last day of mating. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth when the parturition was defined complete (Day 0 post partum).

Oestrous cyclicity (parental animals):

- Stock females: monitored by vaginal smears for 2 weeks before allocation
- After allocation: Vaginal smears were taken in the morning from Day 1 of treatment, up to positive identification of mating including 2 weeks before pairing.

Sperm parameters (parental animals):

Parameters examined in male parental generations.
Determination of the tubular stages of the spermatogenic cycle, such as:
- missing germ cell layers or types,
- retained spermatids,
- multinucleated or apoptotic germ cells and
- sloughing of spermatogenic cells into the lumen.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum
- maximum of 4 pups/litter

PARAMETERS EXAMINED
- Pup identification: As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
- Pup weight: Live pups were individually weighed on Days 1, 4 and 13 post partum.
- Pup observation: once daily for all litters from Day 0 post partum.
- Clinical biochemistry: Blood collection for hormone determination (T4, TSH in serum) was performed only for animals at termination. On Days 4 and 14 post partum, as part of the necropsy procedure, one pooled blood sample per sex (when possible and of approximately 1.0 mL each aliquot) was withdrawn for each litter. Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture). The order of collection was equalised between groups.
- Anogenital distance (AGD): measured on Day 1 post partum. The AGD was normalized to the cube root of body weight collected on Day 1 post partum.
- Nipple count on Day 13 post partum: in each male pup.


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
Parental animals that had completed the scheduled test period were killed by exsanguination under isoflurane anaesthesia. The males were killed after the mating of all females on Day 31 of the study. The females with live pups were killed on Day 14 post partum. Females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. Females, which did not give birth 25 days after positive identification of mating were killed shortly after. Females that did not mate was sacrificed after 30 days from the end of the mating period.

GROSS NECROPSY
Post mortem examinations: including examination of the external surface and orifices. Changes were noted, the requisite organs weighed (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.
o Females: all females were examined also for the following: 1. external and internal abnormalities; 2. external and internal abnormalities;
Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

OGAN WEIGHTS
- From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed: epididymides, kidneys, liver, mesenteric lymph node, ovaries with oviducts, prostate gland (dorsolateral and ventral), seminal vesicles with coagulating glands, spleen, testes, thyroid, uterus – cervix
- The ratios of organ weight to body weight were calculated for each animal.

TISSUE FIXATION
Samples of the following tissues were fixed and preserved in 10% neutral buffered formalin: abnormalities, clitoral gland, epididymides, kidneys, liver, mesenteric lymph node, ovaries with oviducts, Parathyroid glands, penis, prostate gland (dorsolateral and ventral), seminal vesicles with coagulating glands, spleen, testes, thyroid, uterus – cervix, vagina

HISTOPATHOLOGY
- The following tissues were required for histopathological examination: abnormalities, clitoral gland, epididymides, ovaries with oviducts, seminal vesicles with coagulating glands, testes, thyroid, uterus – cervix, vagina.
- After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed. The examination was restricted as described:
o Tissues specified from all males and females in the control and high dose groups killed at term.
o Tissues specified from all animals killed or dying during the treatment period.
o All abnormalities in all groups.
- A detailed qualitative evaluation of testes was performed on all control and high dose males.

Postmortem examinations (offspring):

SACRIFICE
- Pups that had completed the scheduled test period (Day 4 (culled pups) or Day 14 (live pups) post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.


GROSS NECROPSY
- All pups found dead in the cage were examined for external and internal abnormalities.
- All culled pups: external examination.
- All live pups:external abnormalities and sex confirmation by gonads inspection.
- All pups with abnormalities were retained in 10% neutral buffered formalin.


ORGAN WEIGTHS
- Thyroids were weighed from one male and one female pup selected for blood collection for hormone determination and preserved in 10% neutral buffered formalin.
- The thyroid weights were determined after fixation.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopatholgical findings was carried out by means of the non-parametric Kolmogorov Smirnov test. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Reproductive indices:

Male mating index (%) = no. of males with confirmed mating/ no. of males cohabitated x 100
Male fertility index (%) = no.of males which induced pregnancy/ no.of males cohabitated x 100
Female mating index (%) = no. of females with confirmed mating/ no. of females cohabitated x 100
Female fertility index (%) = no.of pregnant females / no.of females cohabitated x 100

Males and Females Pre Coital Interval = The number of nights paired prior to the detection of mating
Pren-natal loss (%) = (no.of visible implantations − live litter size at birth) / no.of visible implantations x 100

Offspring viability indices:

Pup loss at Day 0 post partum (%) = Total litter size − live litter size / total litter size x 100

Post-natal loss at Day 4 post partum (%) = Live litter size at birth − live litter size at Day 4(before culling)/ Live litter size at birth x 100

Post-natal loss at Day 13 post partum (%) = Live litter size on Day 4(after culling) − live litter size at Day 13/ Live litter size on Day 4(after culling) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was the only treatment-related clinical signs recorded in treated animals during the study, affecting both genders.
Animals of the control and low dose group did not show any sign during the whole treatment period.
Some males at mid dose group showed salivation during single days and only in the
mating phase; some females of the same group had salivation during the gestation period.
Most of the animals of the high dose group had salivation for a longer period, during mating phase for males, and during gestation and post partum phases for females.
The number of animals affected and the duration of the signs increased with increasing dose.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1x control male: dead on Day 17 of the mating phase. No clinical signs were noted in this animal before its death. At macroscopic observation, red staining of muzzle and dark red colour lungs were observed; no findings were noted at microscopic observation. The pathological picture observed in this animal did not allow to
establish the cause of death.
1x male low dose group: dead on Day 15 of the premating phase. No clinical signs were noted in this animal before its death. At macroscopic observations, the most relevant findings were dark colour and incomplete collapse of lungs and red
staining of the muzzle. At microscopic observations, alveolar haemorrhage and oedema associated with perivascular presence of yellow material ( test item like) were noted. The cause of death of this animal was attributed to misdosing.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No changes in body weights were noted in males and females treated in low and mid dose group when compared to control group.
Lower body weights, even if not statistically significant and ranging around -5%/-6%, were noted in males of the high dose group, starting from Day 8 of the premating phase up to the end of the study.
The lower body weights were more evident in females in the high dose group being
statistically significant from Day 14 of the gestation period up to Day 4 post partum. The reduction ranged from -8% to -12% and did not recover also during the post partum period.

A decrease in terminal body weight, was observed in high dose males ( -8%) and females (-13%) when compared to the controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No relevant change in food consumption were noted in animals of the low and mid-dose groups during the study, when compared to control.
Males and females of the high dose group showed a slight decrease in food intake on Day 8 of the premating phase.
Females of the high dose group had an overall decreased food consumption during both gestation and post partum phases, being statistically significant decreased only on Day 14 of the gestation period (-14%).

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:

no effects observed

Description (incidence and severity):

No differences between control and treated animals were recorded.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Any microscopic observations had a comparable incidence in control and treated groups
and/or are characteristically seen in untreated rats of the same age and were considered
incidental and unrelated to treatment.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The total number of oestrous cycles observed in all females before pairing (number of non sequential days in which the females were in oestrous) were similar between control and treated groups and was of 3/4 cycles (mean value).

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- The number of copulatory plugs (mean value) found in the cage were 2 in control and 3/2 in the remaining groups. Animals, both in control and treated groups, mated after 3/4 days (mean pre-coital interval) of cohabitation.
- Copulatory index for males and females was comparable between control and treated animals.
- Mean gestation period was comparable between control and treated animals, being of 22/23 days.
- No signs of toxicity were noted in animals in low and mid dose group, when compared to control animals.
- Fertility index of animals dosed at 75 and 225 mg/kg/day was comparable to control animals but it was lower (80%) in males and females dosed at 750 mg/kg/day. This data may suggest a reduced fertility in males and females dosed at 750 mg/kg/day.
- Implantation sites and total litter size at birth were lower in females at the high dose group (-36% and -32%, respectively). Pre-natal loss was 1.6-fold higher in the high dose group compared to control, which was, however, only due to one animal having one pup. These data indicate a treatment-related effect of the test item on female reproductive performance.
- Litter data - Day 0 to Day 4 post partum - before culling: Reductions of total litter size, live litter size and mean litter weight were observed at birth and on Day 1 post partum in females within the high dose group, when compared to controls ( 8.75 versus 12.67 in controls). These reductions ranged between -24% to -32%. Pup loss at birth was higher than control group (13.64% versus 0.86% in controls) which was, however, only due to one animal having one pup. On Day 4, live litter size and post-natal loss was comparable between groups but litter weight was still lower than control group (-19%). However, mean pup weight on Days 1 and 4 was comparable to controls.
- Litter data - Day 13 post partum - after culling: Live litter size and post natal loss was comparable between groups, on Day 13 post partum. Litter weight and mean pup weight, however, showed statistically significant reduction at 750 mg/kg/day, being of -17% for both parameters (mean litter weight: 215.5 g versus 259.8 g in controls; mean pup weight: 26.95 g versus 32.47 g in controls). The above findings showed a treatment-related effect of the test item on the size of litters at birth, on their viability and growth during the post partum period.

Dose descriptor:

NOAEL

Remarks:

(general toxicity)

Effect level:

225 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

Dose descriptor:

NOAEL

Remarks:

(reproductive performance and fertility)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: reduced mating performance, fertility, implantation size, litter size

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Some clinical signs, that are not related to treatment since no correlation can be noted,
were observed in single pups from all groups. These signs were tip of tail black, damaged tail, corneal opacity and one pup was found small, cold to touch and apparently no food intake in the high dose group. Corneal opacity was also observed but it was considered as incidental since noted in single pups and without any relation with the dose.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No differences between control and treated animals were recorded.

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio at birth and on Days 4 and 14 post partum was comparable between treatment and control groups.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Slight statistically significant increases in the anogenital distance (normalised value), performed on Day 1 post partum, were seen between control and male and female pups from dams in the mid dose group. Due to the absence of dose relation and since the data was in the range of historical control values, this finding was considered to be unrelated to treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were retained since not observed in male pups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

no effects in thyroid weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No relevant signs were observed in pups sacrificed on Days 4 and 14 post partum.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mean litter weight and/or pup weight

Reproductive effects observed:

yes

Lowest effective dose / conc.:

225 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Table 1:Changes in mean absolute organ weight (% variation from control)

Sex		Males			Females
Dose (mg/kg/day)		75	225	750	75	225	750
Group		2	3	4	2	3	4
Mesenteric nodes	Abs.	-10%	+38%	*+53%	+7%	+22%	*+69%
	Rel.	-8%	*+45%	*+68%	+6%	+20%	*+86%
* = statistically significant; Abs = absolute, Rel = relative

Table 2: Details of the pregnant status

Groups	1	2	3	4
Initial group size	10	10	10	10
Non pregnant females	1	1	0	2
Did not mate	0	1	0	0
Unilateral implantation	0	0	0	1*
Total litter loss	0	0	0	2
Conceiving (1-5 days)	10	9	8	8
Conceiving (6-14 days)	0	0	2	0
With live pups on Day 14 post partum	9	9	10	6

* The animal was found with Total Litter Loss on Day 0

 

Table 3 Body weight (g) and body weight gain per day (g) of males - Group mean data

♂	Body weight (mean)					BW GAIN°
	Premating		Mating		Sacrifice	Mating	Termination
Group	1	8	15 #/1	8	15	15#	15
1	358.92	376.89	392.57	398.10	412.77	2.240	2.095
2	359.97	376.08	389.81	398.29	414.91	2.022	2.374
3	361.20	375.04	385.81	387.80	400.25	1.539	1.777
4	361.72	359.75	371.82	376.63	387.05	1.725	1.489

#start of mating phase

°= mean daily body weight gain over the previous period starting from the day of allocation

Table 4 Body weight (g) and body weight gain per day (g) of females - Group mean data

♀	Body weight (mean)											BW GAIN°
	Post-coitum			gestation				Post partum
Group	1	8	15#	0	7	14	20	1	4	7	13	#15	Gestation D14	Gestation D20	Post partum D7	Termination
1	240.03	249.65	252.09	255.03	282.91	317.71	387.30	300.77	319.92	322.75	342.87	0.347	4.972	11.599	0.943	3.355
2	236.54	242.45	246.2	250.34	279.40	309.11	379.72	297.67	317.72	322.66	334.91	0.377	4.245	11.768	1.647	2.041
3	243.23	250.32	254.07	259.75	285.70	312.36	388.17	305.04	320.34	325.73	343.08	0.536	3.809*	12.636	1.795	2.892
4	238.80	239.22	245.57	246.13	268.48	291.87** =8,14%	341.53** =11,82%	275.64* =8,36%	290.10** =9,3%	301.86	321.78 =6,16%	0.908	3.341**	8.277*($)	3.818*	3.320

* = mean value of group is significantly different from control at p < 0.05 ** = mean value of group is significantly different from control at p < 0.01 Statistical analysis: Dunnett`s test if group variances are homogeneous Modified t test if group variances are inhomogeneous ($)

° = mean daily body weight gain over the previous period starting from Day 0 post coitum and Day 1 post partum

Table 5 Food consumption (g/animal/day) of pregnant females - gestation period-group mean data

♀	Food consumption (mean)
	Gestation
Group	7	14	20
1	21.74	26.59	27.76
2	22.43	25.77	28.22
3	21.97	25.64	28.85
4	20.02	22.94**	26.43

Table 6 Reproductive Performane 

	Estrous cycle	Mating Index		Implantation Sites (mean)	Fertility Index		At Birth (Mean)		Day13 post partum (Mean)		Prenatal loss %	Gestation length (Mean)
Group	Length (Mean)	Male	Female	Female	Male	Female	Live litter size	Pup loss %	Litter weight (g)	Mean pup weight (g)
1	3	100 %	100 %	13.89	90 %	90 %	12.56	0.86	259.76	32.47	9.10	23
2	4	89 %	90 %	13.22	89 %	90 %	12.22	0.74	260.17	32.98	7.16	22
3	3	100 %	100 %	13.90	100 %	100 %	12.80	1.48	247.27	31.29	7.83	22
4	4	100 %	100 %	8.88*	80 %	80 %	8.50	13.64	215.48*	26.95*	15.03	23
							Total 8.75 *

Statistical analysis(Litter size at birth + Litter weight/pup weight at D13 post partum): Kruskall Wallis test William’s test if group mean differences are different from control at p < 0.05 * = mean value of group is significantly different from control

Statistical analysis (Implantation sites, prenatal loss, gestation length): Kruskall Wallis test William’s test if group mean differences are different from control at p < 0.05 * = mean value of group is significantly different from control

 

Table 7 Absolute organ weights (Mean data)

Male
Organ		Group 1 (N=9)	Group 2 (N=9)	Group 3 (N=10)	Group 4 (N=10)
Mesenteric nodes	Mean	0.4429	0.3999	0.6114	0.6785
	Group diff. at p < 0.05		0.1233	0.1201*	0.1201*
	Group diff. at p < 0.01		0.1565	0.1526*	0.1526*
Thyroid	Mean	0.0243	0.0201	0.0208	0.0226
	Group diff. at p < 0.05		0.0037*	0.0036	0.0036
	Group diff. at p < 0.01		0.0047	0.0046	0.0046
Female
Organ		Group 1 (N=10)	Group 2 (N=10)	Group 3 (N=10)	Group 4 (N=10)
Liver	Mean	13.619	13.468	13.697	11.442
	Group diff. at p < 0.05		2.077	1.414	1.946*
	Group diff. at p < 0.01		2.981	2.029	2.793
Mesenteric nodes	Mean	0.4401	0.4401	0.5384	0.7438
	Group diff. at p < 0.05		0.1738	0.1738	0.2204
	Group diff. at p < 0.01		0.2204	0.2204	0.2204*

Analysis of variance(Male_Mesenteric nodes): F ratio = 14.86 Df = 3/ 34 F probability = 0.000 Note: a * indicates group mean is significantly different from control at level of significance shown.

Analysis of variance(Female_Liver): F ratio = 3.60 Df = 3/ 36 F probability = 0.022 Note: a * indicates group mean is significantly different from control at level of significance shown.

Analysis of variance(Female_Mesenteric nodes): F ratio = 7.46 Df = 3/ 36 F probability = 0.001 Note: a * indicates group mean is significantly different from control at level of significance shown.

Conclusions:

Based on the results of the present study, the test item may be considered toxic for reproduction and development at dose levels of 750 mg/kg body weight/day. The NOAEL (No Observed Adverse Effect Level) for adult fertility and reproduction parameters as well as for litters, was considered to be 225 mg/kg/day for males and females.

Executive summary:

The effect of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring were investigated, when administered daily by oral gavage to males (up to 30 days) and females (from 51 to 57) Wistar Hannover rat. The vehicle was 0.5% carboxymethylcellulose in softened water (0.5% CMC). All doses (75, 225 and 750 mg/kg/day) were administered at a constant volume of 10 mL/kg body weight.

The following investigations were performed on parental animals of all groups: mortality check, clinical signs, body weight, body weight gain, food consumption and mating performance, oestrous cycle evaluation for parental females (2 weeks before dosing, during pre-mating and mating phases, prior to necropsy), thyroid hormone determination only for parental males, litter data, macroscopic observations, organ weights and histopathological examination. Clinical signs, anogenital distance, external and internal examination of pups at necropsy were also recorded. In addition, thyroid hormone levels were determined in pup blood sample/sex, randomly selected at Day 14 post partum. Histopathological evaluation of reproductive organs/tissues was performed on all control and high dose males and females, as well as on all abnormalities detected during post mortem observation.

Mortality

Two cases of premature deaths occurred during the study. One male from control group was found dead on Day 17 of study (during the mating phase) and one male of the low dose group was found dead on Day 15 of the premating phase. No clinical signs were recorded in these two animals before their deaths. It was not possible to identify the cause of death for the control animal while it was established as misgavage for the low dose male.

Clinical signs

Salivation was the only treatment-related clinical signs recorded in treated animals during the study, affecting both genders. This sign was recorded in single occasions in some animals treated at 225 mg/kg/day, during the mating phase. Salivation was recorded for a longer period in most of the animals treated at 750 mg/kg/day, during mating phase for males, and during gestation and post partum phases for females.

Body weight and body weight gain

Lower body weights compared to controls, generally statistically significant, were seen in females dosed at 750 mg/kg/day, starting from Day 14 of the gestation period up to Day 4 post partum. The difference ranged from -8% to -12% and did not recover.

Food consumption

Females of the high dose group had an overall decreased food consumption during both gestation and post partum phases, being statistically significant decreased only on Day 14 of the gestation period (-14%).

Thyroid hormones

Thyroid hormones determination in adult males and male and female pups performed on Day 14 post partum No differences between control and treated animals were recorded.

Oestrous cycle, reproductive parameters, pairing combination and mating performance

Oestrous cycle, pre-coital intervals and copulatory index did not show any differences between groups. Fertility index of males and females dosed at 750 mg/kg/day was found decreased (80%).

Implantation sites, pre-natal loss data and gestation length of females

Mean gestation period was comparable between control and treated animals, being of 22/23 days. Implantation sites were decreased in females dosed at 750 mg/kg/day (8.88 versus 13.89 in controls). Pre-natal loss was higher in the high dose group compared to control (15.03% versus 9.10% in controls).

Litter data at birth, on Days 1 and 4 post partum (before culling) and on Day 13 post partum (after culling) and sex ratio of pups

Litter data

Reductions of litter size (8.75 versus 12.67 in controls) and mean litter weight (215 g versus 260 g in controls) were observed at birth and on Day 1 post partum in females dosed at 750 mg/kg/day, when compared to controls. Pup loss at birth was higher than control group (13.64% versus 0.86% in controls), which was due to one female that lost its only pup. On Day 4, live litter size and post-natal loss was comparable between groups but litter weight was still lower than control group. No alteration in mean pup weight was noted on Days 1 and 4. On Day 13 post partum, live litter size and post natal loss was comparable between groups. Litter weight and mean pup weight, however, showed statistically significant reduction at 750 mg/kg/day, being of -17% for both parameters (mean litter weight: 215.5 g versus 259.8 g in controls; mean pup weigh: 26.95 g versus 32.47 g in controls).

Sex ratio

No significant differences were observed in sex ratio at birth and on Days 4 and 14 post partum.

Clinical signs of pups

No treatment-related clinical signs in pups were noted.

Anogenital distance

No differences in the anogenital distance (normalised value), that can be related to treatment, were noted in pups on Day 1 post partum.

Necropsy findings in decedent pups, in pups sacrificed on Days 4 and 14 post partum and nipple count

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect. No nipples were observed in male pups.

Pups thyroid weight on Day 14 post partum

No significant differences were observed at statistical analysis in the weight of thyroid in treated pups, when compared to controls.

Terminal body weight and organ weights

A slight decrease, statistically significant or not, in terminal body weight was observed in high dose animals of both sexes when compared to the controls. An increase in absolute and relative mesenteric nodes weight, statistically significant or not, was observed in high dose animals of both sexes when compared to the controls.

Macroscopic observations

No remarkable differences were noted at post mortem examination in treated animals, when compared to the controls.

Microscopic observations

No treatment-related changes were observed in treated animals, when compared to the controls. Seminiferous tubules were evaluated with respect to their stage in the spermato[1]genic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Conclusion

In conclusion, possible effects of the test item at the dose level of 750 mg/kg/day on mating performance and fertility in cannot be excluded, considering the decreased fertility index and the reduced number of pregnant females. Only 6 females had live pups at the end of the study. Effects on conception, development of conceptus and parturition were also demonstrated by reductions in implantation sites and total litter size at birth in females dosed at750 mg/kg/day. Mean litter weights and/or mean pup weights were also affected by treatment, being mainly reduced on Day 14 post partum in pups dosed at 750 mg/kg/day. These signs revealed an effect of the test item on the viability and growth of the offspring during the post partum period. These effects were associated with slight in vivo effects on body weight and food consumption on males and especially females treated at 750 mg/kg/day. No adverse effects for general toxicity, reproductive and developmental toxicity were observed in male and female animals dosed at 75 and 225 mg/kg/day. Based on the results of the present study, the test item may be considered toxic for reproduction and development at dose level of 750 mg/kg body weight/day. The NOAEL (No Observed Adverse Effect Level) for adult fertility and reproduction parameters as well as for litters, is 225 mg/kg/day for males and females.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

225 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

A reproductive/developmental toxicity screening test (OECD 421) with the test substance is available.

Data from this study:

The effect of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring were investigated, when administered daily by oral gavage to males (up to 30 days) and females (up to 51 - 57 days) Wistar Hannover rat. The vehicle was 0.5% carboxymethylcellulose in softened water (0.5% CMC). All doses (75, 225 and 750 mg/kg bw/day) were administered at a constant volume of 10 mL/kg body weight.

Two cases of premature deaths occurred during the study. One male from the control group was found dead on Day 17 of the study (during the mating phase) and one male of the low dose group was found dead on Day 15 of the premating phase. No clinical signs were recorded in these two animals before their deaths. It was not possible to identify the cause of death for the control animal while it was established as misgavage for the low dose male.

Salivation was the only treatment-related clinical signs recorded in treated animals during the study, affecting both genders. This sign was recorded in single occasions in some animals treated at 225 mg/kg bw/day, during the mating phase. Salivation was recorded for a longer period in most of the animals treated at 750 mg/kg bw/day, during mating phase for males, and during gestation and post partum phases for females.

Lower body weights compared to controls, generally statistically significant, were seen in females dosed at 750 mg/kg bw/day, starting from Day 14 of the gestation period up to Day 4 post partum. The difference ranged from -8% to -12% and did not recover.

Females of the high dose group had an overall decreased food consumption during both gestation and post partum phases, being statistically significant decreased only on Day 14 of the gestation period (-14%).

Thyroid hormones determination in adult males and male and female pups performed on Day 14 post partum. No differences between control and treated animals were recorded.

Oestrous cycle, pre-coital intervals and copulatory index did not show any differences between groups. Fertility index of males and females dosed at 750 mg/kg bw/day was found decreased (80%).

Mean gestation period was comparable between control and treated animals, being of 22/23 days. Implantation sites were decreased in females dosed at 750 mg/kg bw/day (8.88 versus 13.89 in controls). Pre-natal loss was higher in the high dose group compared to control (15.03% versus 9.10% in controls).

Reductions of litter size (8.75 versus 12.67 in controls) and mean litter weight (215 g versus 260 g in controls) were observed at birth and on Day 1 post partum in females dosed at 750 mg/kg/day, when compared to controls. Pup loss at birth was higher than control group (13.64% versus 0.86% in controls), which was due to one female that lost its only pup. On Day 4, live litter size and post-natal loss was comparable between groups but litter weight was still lower than control group. No alteration in mean pup weight was noted on Days 1 and 4. On Day 13 post partum, live litter size and post natal loss was comparable between groups. Litter weight and mean pup weight, however, showed statistically significant reduction at 750 mg/kg bw/day, being of -17% for both parameters (mean litter weight: 215.5 g versus 259.8 g in controls; mean pup weigh: 26.95 g versus 32.47 g in controls).

No significant differences were observed in sex ratio at birth and on Days 4 and 14 post partum.

No treatment-related clinical signs in pups were noted.

No differences in the anogenital distance (normalised value), that can be related to treatment, were noted in pups on Day 1 post partum.

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect. No nipples were observed in male pups.

No significant differences were observed at statistical analysis in the weight of thyroid in treated pups, when compared to controls.

A slight decrease, statistically significant or not, in terminal body weight was observed in high dose animals of both sexes when compared to the controls. An increase in absolute and relative mesenteric nodes weight, statistically significant or not, was observed in high dose animals of both sexes when compared to the controls.

No remarkable differences were noted at post mortem examination in treated animals, when compared to the controls.

No treatment-related changes were observed in treated animals, when compared to the controls. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

In conclusion, possible direct effects of the test item at the dose level of 750 mg/kg bw/day on mating performance and fertility cannot be excluded, considering the decreased fertility index and the reduced number of pregnant females. Only 6 females had live pups at the end of the study. Effects on conception, development of conceptus and parturition were also demonstrated by reduced implantation sites and total litter size at birth in females dosed at 750 mg/kg bw/day. Mean litter weights and/or mean pup weights were also affected by treatment, being mainly reduced on Day 14 post partum in pups dosed at 750 mg/kg bw/day. These signs revealed an effect of the test item on the viability and growth of the offspring during the post partum period. These effects were associated with slight effects on body weight and food consumption on males and especially females treated at 750 mg/kg bw/day. In addition, the mesenteric lymph node weight was increased in the high (+29%) and mid dose (+55%) group. Increased mesenteric lymph node weight was also observed in a 90 day-repeated dose toxicity study (OECD 408: Ciba-Geigy. 874234. 1989). The effect already occurred at dose levels of 47.9 mg/kg bw/day and was accompanied by foam cells and inflammatory and/or necrotizing changes in mesenteric lymph nodes, which presents severe suffering for the tested animals. As systemic toxicity can influence effects seen on reproductive parameters, these findings have to be further clarified in the ongoing extended-one generation study (OECD 443). Besides this, no adverse effects for general toxicity, reproductive and developmental toxicity were observed in male and female animals dosed at 75 and 225 mg/kg bw/day in the conducted OECD 421 study.

Based on the current results from the OECD 421 study, the test item may be considered toxic for reproduction and development at dose level of 750 mg/kg bw/day. The NOAEL (No Observed Adverse Effect Level) for adult fertility and reproduction parameters as well as for pups, is 225 mg/kg bw/day for males and females.

 

Effects on developmental toxicity

Description of key information

The OECD 421 was not part of the requests concerning the Final Decision of a Compliance Check CCH-D-2114476180-53-01/F. However, the study was performed to provide sufficient information on development and reproduction in order to strengthen a read-across approach to a structural analogue substance. If the data would not support the read-across justification, the registrant would perform the requested OECD 414 studies in both species. This strategy was decided during a phone call with ECHA representatives.

The results of a reproduction/developmental toxicity screening study (OECD 421) with the test item in rats by the oral route indicate that the test substance may be considered toxic for reproduction and development at high doses. The NOAEL for adult fertility and reproduction parameters as well as for pups was set to 225mg/kg bw/day for males and females. The obtained data from the OECD 421 study did not strengthen the provided read-across justification. As the significance of the findings for human health is not clear at this time, a developmental toxicity study (OECD 414) with the test substance in rats by the oral route was perforned. Results indicate, that the potential to cause toxicity to development cannot be excluded when given in high doses. The NOAEL for developmental toxicity as well as for maternal toxicity was 225 mg/kg bw/day.

A teratogenicity study (OECD 414) with the test substance in a second species (rabbit) is ongoing. 

In addition, an Extended-One-Generation study (OECD 443) is ongoing. An update of all relevant information will be submitted at the earliest date possible but not earlier than Q3 2023.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 9 weeks (females), 11 weeks (males)
- Weight at study initiation: 201-241 g for virgin females, at least 328 g for males
- Housing: no more than 5 of one sex before and after mating in polysulfone solid bottomed cages, measuring 59.5×38×20cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided. During mating, 1 male and 1 female were kept in polysulfone cages measuring 42.5×26.6×18.5cm with a stainless steel mesh lid and floor Tecniplast Gazzada S.a.r.l., Buguggiate, Varese).
- Diet: laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy) with certified dietary levels of phytooestrogen (genistein less 350 μg), ad libitum
- Water: drinking water ad libitum
- Acclimation period: 26 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2
- Humidity (%): 55±15
- Photoperiod (hrs dark / hrs light): 12 / 12
- Air changes/ hour: 15-20

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of powder of the test item was weighed (correcting for purity) and then transferred to a mortar and with the aid of pestle, the granularity of the test item was reduced to a small size; a small amount of the vehicle was added to the powder and mixed to forma smooth
paste; the paste was transferred to a suitable container and rinsed the mortar with additional
vehicle (at least 3×); the washings was transferred to the main container and diluted to volume with vehicle; the obtained suspensions was then homogenised (by Silverson) for approximately
5 minutes and left overnight on a magnetic stirrer (for at least 16 hours) at room temperature prior to use and up until the time of dosing of the last animal.

DIET PREPARATION
- Rate of preparation of diet (frequency): daily or weekly

VEHICLE
- Concentration in vehicle: 0.5%
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- Females were paired overnight, 1:1
- the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0.

Duration of treatment / exposure:

GD 6-19

Frequency of treatment:

once daily

Duration of test:

on GD 20, all surviving females were sacrificed

Remarks:

Doses / Concentrations:
75, 225 and 750 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selection of the dose levels of 75, 225 and 750 mg/kg bw/day was based on information from a preliminary non-GLP compliant study
- The oral route was selected as it is a possible route of exposure of the test item in man
- The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day on working days, weekends and Public Holidays throughout the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day for individual animals

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 3, 6, 9, 12, 15, 18 and 20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: GD 3, 6, 9, 12, 15, 18 and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Brain, mesenteric lymph node, spleen, thyroid
- Tissue Fixation: Mesenteric lymph node, spleen, thyroid
- Histophathology: Abnormalities, Thyroid

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number, sex and weight of all live foetuses, internal foetal sex determination in each foetuses allocated to the skeletal examination, anogenital distance (AGD) in all live foetuses, number of intra-uterine deaths, gross evaluation of placentae

Blood sampling:

- Serum: Yes
- Volume collected: 0.8 mL of blood samples. The serum obtained was divided in two aliquots (first with 250 µL and the second with the remaining amount)

Fetal examinations:

- Weight of each fetus: Yes
- Sex: Yes
- Gross evaluation of placentae: Yes
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter

Statistics:

- Dunnett’s test or a modified t test: for continous variables
- Kruskal-Wallis test: for non-continuous variables
- non-parametric version of theWilliams test: for intergroup differences between the control and treated groups
- The criterion for statistical significance was p<0.05. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.

Indices:

Pre-implantation loss % = no. of corpora lutea−no. of implantations/no. of corpora lutea x 100

Post-implantation loss % = no. of implantations−no. of live foetuses/ no. of implantations x 100

Total implantation loss % = (no. of corpora lutea−no. of live foetuses)/no. of corpora lutea x 100

Details on maternal toxic effects:

- MORTALITY:
No mortality occurred.

- CLINICAL SYMPTOMS:
Piloerection was the principal treatment-related clinical sign noted in females receiving 750 mg/kg bw/day. The sign appeared after approximately one week of treatment within 15-30 minutes of dosing. In some cases it was persisted and then recorded also before dosing. In addition, six females showed hunched posture during the second part of the gestation period for a maximum of six consecutive days. Hairloss was noted in two females. Scabs (one female) and hairloss on head (4 females) were evident at 225 mg/kg bw/day. With the exception of one female in which these signs occurred simultaneously (from Day 11 post coitum onwards). In the other case hairloss were sporadically observed. Scabs and hairloss on head were noted in one female for three consecutive days (Days 18 to 20) dosed at 75 mg/kg bw/day.

- FOOD CONSUMPTION:
Food consumption was statistically significant decreased in females receiving 750 mg/kg bw/day from Days 9 to 20 post coitum (-26 % to -37 % when compared with controls). Compared to the control and significant, at statistical analysis, a reduction in food consumption was also seen from Days 9 to 15 post coitum at 225 mg/kg bw/day (-11 % to -19 %) and limited to Day 12 in females receiving 75 mg/kg bw/day (-11%).

- BODY WEIGHT:
A slight, but statistically significant decrease in mean body weights (not greater than 10%, compared to the controls), was observed between Days 12 to 20 post coitum in females receiving 750 mg/kg bw/day.
Reduced body weight gain was consistently noted at 750 mg/kg bw/day, from Days 9 to 20 post coitum, with the significance at statistical analysis evident (-32% to -88%). Compared to the control group the biggest difference was seen on Day 9 post coitum (-88%). This trend indicated a maternal toxicity at this dose level. No relevant differences in body weight and body weight gain were noted at the dose levels
of 225 and 75 mg/kg bw/day.
- TERMINAL BODY WEIGHT, CORRECTED BODY WEIGHT AND BODY WEIGHT GAIN:
Terminal body weight and corrected maternal body weight were statistically significant decreased in females dosed at 750 mg/kg bw/day, when compared to the control group (not greater than -11%). The effect on maternal body weight loss was better evident in the corrected body weight gain (terminal body weight at necropsy minus gravid uterine weight, minus body weight at Day 6 post coitum) which was decreased (significant, at statistical analysis) in females of the 750 mg/kg bw/day group. No changes were noted in the lower dose levels tested. The decrease in corrected body weight gain noted at 225 mg/kg bw/day was without significance, at statistical analysis, it was dose-unrelated and then considered to be fortuitous (they were ascribed to body weight losses observed in three females).

- THYROID HORMONE:
No differences between control and treated animals were recorded. T4 values were slightly, but statistically significantly lower at 750 mg/kg bw/day. Based on the minimal difference and in absence of any other differences (T3, TSH, thyroid weight and histopathology), this is not considered to be toxicologically relevant.
- NECROPSY FINDINGS:
> ORGAN WEIGHTS: Statistically significant increase in absolute and relative mesenteric nodes weight was observed in females at 750 mg/kg bw/day (+29 % relative to brain weight) and 225 mg/kg bw/day (+53 % relative to brain weight) when compared to the controls. Any organ weight changes other than those listed above were within the range of occasionally observed and expected spontaneous changes in rats of the same age and considered unrelated to treatment.
> MACROSCOPIC OBSERVATIONS: Treated females did not show relevant macroscopic changes. Any macroscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.
> MICROSCOPIC OBSERVATIONS: No treatment-related changes were noted in thyroid as well as on all abnormalities. Any microscopic observations had a comparable incidence in control and treated groups
and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

- REPRODUCTION DATA:
Refer to Table 1 (Section: Any other information on results incl tables)

Dose descriptor:

NOAEL

Effect level:

225 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: Maternal toxicity: Clinical signs (piloerection, hunched posture), reduced (corrected) body weight & (corrected) body weigt gain & terminal body weight & food consumption, increased mesenteric lymph node weight (abs/rel)

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

- LITTER DATA: All reproductive outcomes of treated females, including the number of corpora lutea, number of implantations sites, live foetuses or uterine deaths, as early and late resorptions, pre-implantation loss and in the sex ratio of the fetuses did not show significant differences against the control. Higher post-implantation loss (mostly due to the loss at early stage of development) was noted at all dose levels when compared to the control groups. However in view of that the values were well within the range of our historical control data and limited in magnitude, the difference was not considered to be toxicologically relevant. Moreover, litter weight and mean foetal weight (for each sexes, as well as for both sexes combined) of treatment groups were comparable to the control group.

- ANOGENITAL DISTANCE: No relevant differences were noted in the mean values of the AGD (anogenital distance normalised for the cube root of the body weight performed on Day 20 post coitum) of foetuses of both sexes maternally exposed at all dose levels compared to the control group.

- EXTERNAL EXAMINATIONS:
At the external examination of the foetuses minor alterations were noted as follows: control group: 4 foetuses small in size (body weight lower than 2.7 g) in 3 litters; 75 mg/kg bw/day: 2 foetuses small in size in 2 litters; 225 mg/kg bw/day: 5 foetuses small in size in 5 litters . Two foetuses in one litter showed abnormal shape of the hindlimbs; 750 mg/kg bw/day: 8 foetuses small in size in 6 litters. An increase incidence in percentage of foetuses and litters affected were noted in the two upper dose levels, when compared to the control group. However the differences of the percentages found between the dose level of 225 and 750 mg/kg/day were low of magnitude. This indicated a poor correlation between dose levels and the anomaly although the incidences of this finding were above our background range.
Major findings included: umbilical hernia in one control foetus, small in size (female no. 1). Multiple alterations like as umbilical hernia, polydactyly and subcutaneous oedema (generalised) was noted in one foetus (female no. 53) maternally exposed at 75 mg/kg bw/day. The occurrence of these major findings in foetuses were considered incidental.

- FIXED VISCERAL EXAMINATIONS:
There was no visceral malformation associated with treatment with the test item at any dose levels.
Two control foetuses in two different litters showed major abnormalities: one (female no. 1) as confirmation of that observed during the external examination of the foetus (umbilical hernia) and the other (female no. 43) on the urinary system including an extreme pelvic dilatation of the kidney (unilateral) associated to the extreme dilatation of the ureter (unilateral).
Overall the minor alterations (variations and anomalies) recorded on the kidneys and ureters (e. g. pelvic dilatation and/or ureters enlarge, slight to moderate), on the testis (displaced) or on the abdominal and thoracic cavities (haemorrhagic) occurred with similar incidence between control and treated groups.
Consequently, under the conditions described for this study the test item did not reveal teratogenic potential up to and including the dose level of 750mg/kg bw/day.

- SKELETAL EXAMINATIONS: Major abnormalities, that mainly included defects in the ossification of the rib, sternebrae and thoracic vertebrae, were recorded in foetuses maternally exposed at 75 (3 foetuses in 3 litters), 225 (2 foetuses in one litter) and 750 mg/kg bw/day (one foetus in one litter) as well as in the control group (one foetus in one litter). In particular, the malformations were related to the absence of one rib in three foetuses, one control (female no. 1) and two (female nos. 59, 77) at 75 mg/kg bw/day. In addition one foetus maternally exposed at 75 mg/kg bw/day (female no. 53) showing polydactyly at
external examination, have one additional metatarsal in the hindpaw, at skeletal examination. Ribs fused and thoracic haemivertebrae were seen in the same foetus at 225 mg/kg bw/day. Cleaved sternum was found in one foetus at 750 mg/kg bw/day. The incidence of malformations were not dose related, suggesting no conclusive correlation with the treatment.
However, the skeletal assessment revealed instances of an increase incidence of irregular sternebral ossification in the high dose foetuses (750 mg/kg bw/day) compared to control. These included ossification findings in the sternal elements as asymmetrical cleaved, bipartite, bifurcate distally or with additional ossification site. Development and ossification of the sternum occurs also in the perinatal period and the alterations noted can be considered as a transient delay of ossification that will resolve shortly after birth rather than as teratogenicity sign. Finally these foetal alterations occurred at the dose level of 750 mg/kg bw/day where maternal toxicity was evident.

Dose descriptor:

NOAEL

Effect level:

225 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

increase incidence of irregular sternebral ossification (transient delay)

Developmental effects observed:

yes

Lowest effective dose / conc.:

225 mg/kg bw/day

Treatment related:

no

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

no

Table 1: Reproductive data

Group (mg/kg bw/day)	1(0)	2 (75)	3 (225)	4 (750)
Unilateral implantation	0	0	0	1
Not pregnant	0	0	2	0
Pregnant females at term	25	25	23	25

 

Table 2: Postimplantation loss

Groups (mg/kg/day)	Group 1 0 mg/kg/day	Group 2 75 mg/kg/day	Group 3 225 mg/kg/day	Group 4 750 mg/kg/day	HCD mean
Post implantation loss	0.51	1.81	2.49	2.40	3.71

 

Table 3: External malformation of foetuses

		Group 1 0 mg/kg/day		Group 2 75 mg/kg/day			Group 3 225 mg/kg/day		Group 4 750 mg/kg/day		HCD mean
Number of foetuses examined		285		281			260		272		849
Number of litter examined		25		25			23		25		70
Incidence of foetuses/litter with		N	%	N	%	N		%	N	%	N	%
Anomalies
Small foetuses Body weight <2.7g	Foetus Litter	4 3	1.4 12.0	2 2	0.71 8.0	5 5		1.9 21.7	8 6	2.9 24.0	4 4	0.5 5.7

HCD = data from 3 studies (OECD 414 inWistar rats) conducted at the Test Facility in years 2014-2018.

 

Table 4: Skeletal examinations of foetuses

		Group 1 0 mg/kg/day		Group 2 75 mg/kg/day		Group 3 225 mg/kg/day		Group 4 750 mg/kg/day
Number of fetuses examined		149		147		136		140
Number of litter examined		25		25		23		25
Incidence of fetuses/litter with		N	%	N	%	N	%	N	%
Malformation
Rib absent	Foetus Litter	1 1	0.67 4	2 2	1.36 8	0 0	0 0	0 0	0 0
Rib fused	Foetus Litter	0 0	0 0	0 0	0 0	1 ^a 1	0.74 4	0 0	0 0
Thoracic Haemivertebrae	Foetus Litter	0 0	0 0	0 0	0 0	1 ^a 1	0.74 4	0 0	0 0
Hindpaw Additional metatarsal	Foetus Litter	0 0	0 0	1 1	0.67 4	0 0	0 0	0 0	0 0
Cleaved sternum	Foetus Litter	0 0	0 0	0 0	0 0	0 0	0 0	1 1	0.71 4

^a Same foetus

Conclusions:

Based on the results obtained in the study, the NOAEL for maternal toxicity and for developmental toxicity could be set at 225 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

225 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

A teratogenicity study with the test substance is available.

Data from this study:

The effects of the test item were investigated in rat during pregnancy and embryo-foetal development. Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each. All females were administered by oral gavage during the gestation period from Day 6 through Day 19 post coitum at 75, 225 and 750 mg/kg bw/day and 10 mL/kg body weight as the dose volume. Control females received the vehicle (0.5% carboxymethylcellulose in softened water (0.5% CMC)) at the same dose volume during the same treatment period.

No mortality of females occurred during the study. The final number of pregnant females on Day 20 post coitum was 25 in the control group, low dose (75 mg/kg bw/day) and top dose (750 mg/kg bw/day) and 23 in the mid dose group (225 mg/kg bw/day).

Piloerection was the treatment-related clinical sign noted in all females receiving 750 mg/kg bw/day. Other clinical sign, such as hunched posture was also noted. No other relevant clinical signs were noted in females receiving 75 and 225 mg/kg bw/day.

Reduction in maternal body weight (not greater than 10%) was observed at 750 mg/kg bw/day between Days 12 to 20 post coitum, when compared to the control group. The maternal growth retardation was better evident on body weight gain which was decreased from Days 9 to 20 post coitum, with significance, at statistical analysis, when compared to the control group.

Statistically significant reduction in food consumption was noted in the high-dose females receiving 750 mg/kg bw/day starting from Day 9 post coitum until termination. Some differences (decrease) observed in the remaining treated groups against to control, was transient and with a trend of recovery.

There was no treatment-related effect on thyroid hormone determination. A decrease in body weight gain, corrected for gravid uterus weight, terminal body weight and body weight at Day 6 post coitum was recorded in treated females receiving 750 mg/kg bw/day (approximately 20%). Terminal body weight and corrected maternal body weight were also statistically significant decreased in females dosed at 750 mg/kg bw/day, when compared to the control group.

Litter data, mean foetal weight and sex ratios did not show relevant differences between control and treated groups.

Compared to the control, no alterations were seen in the anogenital distance performed in foetuses maternally exposed to the test item.

Statistically significant increase in absolute and relative mesenteric nodes weight was observed in females receiving the dose levels ≥ 225 mg/kg bw/day, when compared to the controls.

Females that completed the treatment period did not show relevant macroscopic changes.

No treatment-related changes were noted in thyroid when examined microscopically.

The overall incidences of foetuses or litters with findings did not indicate test item-related effect.

No relevant changes that could be considered treatment-related were observed at visceral examination of foetuses between the control and treated groups. Consequently, under the conditions described for this study, the test item did not reveal teratogenic potential up to and including the dose level of 750mg/kg bw/day.

The skeletal assessment revealed instances of an increase incidence of irregular sternebral ossification in the high dose foetuses (750 mg/kg bw/day) compared to control. The alterations  noted can be considered as a transient delay of ossification that will resolve shortly after birth rather than as teratogenicity sign.

On the basis of the results obtained in this study, it could be concluded that the test item induce signs of maternal toxicity and developmental toxicity at dose level of 750 mg/kg bw/day. Therefore, the NOAEL for maternal toxicity and for developmental toxicity is set at 225 mg/kg bw/day.

Justification for classification or non-classification

Classification, Labeling, 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 considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.