Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 March 2020 to 07 August 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Cetrimonium chloride
EC Number:
203-928-6
EC Name:
Cetrimonium chloride
Cas Number:
112-02-7
Molecular formula:
C19H42N.Cl
IUPAC Name:
N,N,N-trimethylhexadecan-1-aminium chloride
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Animal supply and acclimatisation
A total of 120 Crl:CD Sprague Dawley (SD) virgin female rats, 9 weeks old (200-225 g) were supplied and received from Charles River Italia S.p.A., Calco (Lecco), Italy. A first batch of 100 females arrived on March 5, 2020 and a second batch of 20 females arrived on March 12, 2020. The male rats used for mating were from the same supplier, and were at least 11 weeks old (at least 350 g). After arrival the weight range was determined and the females were uniquely identified by tattoo on the hind feet. A health check was then performed by a veterinarian. An acclimatisation period of 18 days for the first batch of animals and 11 days for the second batch was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

Animal husbandry
The rats were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C 2°C and 55% 15%, respectively; actual conditions were monitored, recorded and the records retained. No deviations from these ranges were recorded during the study. There were approximately 15-20 air changes per hour and the rooms were lit by artificial light for 12 hours each day. Before mating for all animals and after mating for males, the animals were housed no more than 5 of one sex to a cage in clear polysulfone cages measuring 59.5 38 20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese); nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week. During the mating period, one male rat was housed with one female rat in clear polysulfone cages measuring 42.5 26.6 18 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese) with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the mated females were housed individually in clear polysulfone cages meas- uring 42.5 26.6 18 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese); nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week. The certificate of analysis of the nesting material used was kept with study raw data. Drinking water was supplied ad libitum to each cage via water bottles. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at ERBC. The certificate of the diet used was kept with study raw data. Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
softened water (by reverse osmosis)
Details on exposure:
All animals were administered the test substance by oral gavage during the gestation period, starting from Day 3 through Day 19 post coitum at the doses of 0, 18.75, 37.5, 75 mg/kg bw/day (25 animals in each group).

Allocation to groups
On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the groups by computerised stratified randomisation (SOP ANI/105) to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed individually. The cages were identified by a label recording the study number, animal number and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in ERBC Study no. A3762 in the range from 1 to 10 mg/mL. The proposed preparation procedure for the test substance was checked in the range from 1 to 10 mg/mL by chemical analysis (concentration and homogeneity) in ERBC Study no. A3762 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in ERBC SOPs for concentration (85-115%) and homogeneity (CV < 10%). In the same study (ERBC Study no. A3762) and in the same range of concentration, the stability of the preparations was verified and the final results were as follows: (a) at 28 hours at room temperature (b) at 10 day stability at 2-8°C. Samples of the preparations prepared on Week 1 and Last week of treatment were analysed to check the homogeneity and concentration. Chemical analysis was carried out by the Analytical Chemistry Department. Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (85-115% for concentration and CV <10% for homogeneity). The validated software used for this activity was Empower® 2 Build No. 2154.
Details on mating procedure:
Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records were maintained.
Duration of treatment / exposure:
All animals were dosed once a day from Day 3 through Day 19 post coitum
Frequency of treatment:
Once a day
Doses / concentrations
Remarks:
0, 18.75, 37.5, 75 mg/kg bw/day
The required amount of test substance was suspended in the vehicle. The preparations were made at up to 8- day intervals (concentrations of 1.875, 3.75 and 7.5 mg/mL). Concentrations were calculated and expressed in terms of test substance corrected for purity.
No. of animals per sex per dose:
Each group comprised 25 mated female rats
Control animals:
yes

Examinations

Maternal examinations:
Throughout the study, all animals were checked for mortality early in the morning and again 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. All clinical signs were recorded for individual animals. Each animal was observed daily and any clinical signs recorded starting from allocation until sacrifice. All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum. Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum. On Day 20 post coitum, blood samples for thyroid hormones determination (T3, T4 and TSH) (approximately 0.5 mL), were collected, randomizing (equalised) between treatment groups, from the sublingual vein of all females, under slight isoflurane anaesthesia. Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA). As a part of the terminal sacrifice procedure, blood samples for haematological investigations were withdrawn from the abdominal vena cava, of all females, under isoflurane anaesthesia, and collected in tubes with EDTA anticoagulant.

All animals were killed by exanguination under isoflurane anaesthesia on Day 20 post coitum. All animals were subjected to necropsy, supervised by a pathologist. From all animals completing the scheduled test period, the thyroid was dissected free of fat, fixed and preserved in 10% neutral buffered formalin. The thyroid weight was determined after fixation. The ratio of thyroid weight to body weight was calculated for each animal. After dehydration and embedding in paraffin wax, sections of the thyroid tissue were cut at 5 micrometre thickness and stained with haematoxylin and eosin. Sections were examined for evaluation of pathological changes in all groups.
Ovaries and uterine content:
The ovaries and uteri were examined to determine:
– Gravid uterine weight;
– number of corpora lutea;
– number of implantation sites;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.

Intra-uterine deaths were classified as:
– early resorptions: only placental remnants visible.
– late resorptions: placental and foetal remnants visible.

Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.
Fetal examinations:
All live foetuses were examined externally. In addition, the anogenital distance (AGD) was recorded in all male and females foetuses. Approximately one-half of the foetuses (i.e., routinely, every second live foetus) in each litter were preserved in Bouin’s solution for subsequent fixed-visceral examination. The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal (single staining) examination. Skeletal and fixed-visceral examinations were performed in all groups. In both cases, sex was confirmed by internal inspection of the gonads.

Structural deviations were classified as follows:

Malformations
Major abnormalities that are rare and/or affect the survival or health of the species under investigation.

Anomalies
Minor abnormalities that are detected relatively frequently.

Variants
A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.
Statistics:
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 non-continuous variables was carried out by means of the Kruskal- Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Indices:
Group mean values for body weight and food consumption of pregnant females, gravid uterus weight, organ weight, absolute weight gain (terminal body weight minus body weight at Day 0 post coitum minus gravid uterus), litter size, intra-uterine deaths, corpora lutea count, number of implantations, total implantation loss, pre- and post-implantation loss, sex ratio, anogenital distance, haematology and thyroid hormone determination were calculated. Data from non-pregnant animals were not included in group mean calculations of maternal body weight.

Sex ratios of the foetuses were calculated as the percentage of males. The anogenital distance (AGD) was calculated as normalized to the cube root of body weight measured on Day 20 post coitum. All derived values (e.g., means, percentages, ratios) first were calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.
Historical control data:
The AGD values range of laboratory historical control data were 1.70 - 3.74 mm/g1/3 for males and 0.96 - 3.00 mm/g1/3 for females.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was the treatment-related clinical sign observed in all treated groups with a dose related incidence: 6 out 25 in Group 2 (18.75 mg/kg bw/day), 17 out of 25 in Group 3 (37.5 mg/kg bw/day) and 23 out of 25 in Group 4 (75 mg/kg bw/day). In Group 4 (75 mg/kg bw/day) this sign was accompanied by hunched posture in a few females. The onset of these signs was on Day 5 post coitum in Group 4 and on Day 7 post coitum in Groups 2 and 3. Minor signs, such as hairloss and damaged ear were sporadically recorded during the study and were considered as incidental.
Mortality:
no mortality observed
Description (incidence):
No animals died during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease (up to -6%) in body weight was observed in females receiving 75 mg/kg bw/day (Group 4) on Days 9, 12 and 20 post coitum. Body weight gain was statistically significantly reduced in Group 4 relative to the control group on two occasions, Days 6 and 20 post coitum. However, values between Days 9 and 18 were comparable to control. Body weight and body weight gain in females receiving 18.75 mg/kg bw/day (Group 2) and 37.5 mg/kg bw/day (Group 3) were comparable to the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduction in food consumption was observed in females receiving 75 mg/kg/day (Group 4) starting from Day 6 post coitum (approximately -29%) through Day 15 post coitum (approximately -8%). No differences were recorded on Day 18 post coitum and again a slight decrease was recorded on Day 20 post coitum (approximately -20%). No differences in food consumption were noted in females of Group 3, compared to controls. The statistically significant decrease (approximately -7%) in food consumption noted in females of Group 2 on one occasion was not considered of toxicological relevance.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
An increase of polychromatophil erythrocytes was observed in the peripheral blood smears of a number of animals of all treated groups. This finding was dose-related in the incidence. Due to the dose-relation observed between groups, the relation with the test item cannot be definitively excluded, even if there were no indications of an effect of the test item on the bone marrow or other tissues/organs. Considering that: (a) no haematological findings were observed (e.g. anaemia) (b) no changes were recorded during the in-vivo phase or at post-mortem examination (c) no treatment-related effects were observed in previous studies (https://echa.europa.eu/it/registration-dossier/-/registered-dossier/14219/7/6/2), the increase of polychromatophil erythrocytes alone was considered to be not adverse.

In addition, monocytes group mean data were statistically significantly higher than controls in females dosed at 75 mg/kg/day (30%). Since the increase of monocytes is usually associated with chronic inflammation/necrosis and no related findings were recorded (e.g. lymphocytosis), and similar monocytes values were found in two control females, the above increase of monocytes was considered to be incidental.

No differences in haematology parameters were noted in females receiving 18.75 mg/kg/day (Group 2) and 37.5 mg/kg/day (Group 3), compared to controls.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No differences between control and treated females were recorded in thyroid hormone.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was no effect on the thyroid weight at any dose levels, compared to the control group.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in thyroid gland of females receiving Cetrimonium chloride at low, medium and high dose.
Details on results:
Statistically significant reductions in terminal body weight (approximately -6%) and gravid uterus weight (-11%), associated with reduction (-29%) in absolute weight gain (terminal body weight minus body weight at Day 0 post coitum minus gravid uterus), were seen in females receiving 75 mg/kg/day (Group 4), compared to controls. No differences in these parameters were noted in females receiving 18.75 mg/kg/day (Group 2) and 37.5 mg/kg/day (Group 3), compared to controls.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No differences in the uterine deaths (early, late and total) were observed.
Details on maternal toxic effects:
No mortality occurred during the study. During the in-life phase, maternal toxicity was noted in females receiving 75 mg/kg bw/day, as indicated by the clinical signs and the reduction in the absolute weight gain. The slight and occasionally decrease of body weight, body weight gain,food consumption, terminal body weight, gravid uterus weight and mean foetal weight were not considered of toxicological relevance since all these findings were of low magnitude and did not affect the pregnancy and developmental outcomes thus, they were considered not adverse. At macroscopic observations, no relevant changes that could be considered treatment- related were recorded. Polychromasia detected at haematology investigation was considered to be not adverse, even though the relation with the test item cannot be excluded. No differences between control and treated females were recorded in thyroid hormones determination, thyroid weight and thyroid histopathology. No difference considered treatment related was noted in the mean values of the anogenital distance of foetuses of both sexes maternally exposed at all dose levels compared to the control group. On the basis of the above results, it can be concluded that the dosage of 75 mg/kg bw/day induced slight maternal toxicity with no adverse effect on pregnancy. The dosages of 37.5 mg/kg bw/day and 18.75 mg/kg bw/day were well tolerated. The NOAEL for maternal toxicity was considered to be 75 mg/kg bw/day.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 75 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: all parameters
Remarks on result:
other: only slight maternal toxicity with no adverse effect on pregnancy
Remarks:
the effects were either toxicologically not relevant (e.g., decrease of body weight, body weight gain, food consumption, terminal body weight, gravid uterus weight and mean foetal weight) due to their low magnitude of change

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease was noted in the mean foetal weight, for each sex (approximately -4 and -5% for females and males respectively), as well as for both sexes combined (approximately-4%). However, considering that the value were within the range of our historical control data and considering dering the limited magnitude of the difference, the finding was not considered to be toxicologically relevant.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 14 small foetuses (< 2.7 g) were detected, 9 out of 356 in the control group, 1 out of 322 in the low dose group, 1 out of 354 in the mid-group and 3 out of 346 in the high dose group. One foetus in the mid-dose group had alterations classified as malformations: imperforate anus and absence of tail (acaudia).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations were detected in control and in treated groups without dose relationship, in terms of foetuses and litters affected, as reported in attached results tables in attached background material section of the IUCLID. The other alterations (anomalies and variations) were seen in treated and control foetuses with similar incidence and/or without dose relationship. The type and distribution of mentioned findings, both for major and minor alterations, were considered incidental and not treatment-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations were detected in mid- and high dose groups as reported in attached results tables in attached background material section of the IUCLID. A total of two foetuses showed malformations: heart ventricle enlarged extreme was observed in one foetus maternally exposed at 37.5 mg/kg bw/day (Group 3) and extremely enlarged ureter in association with kidney pelvic dilatation extreme in one foetus maternally exposed at 75 mg/kg bw/day (Group 4). These findings were considered incidental since they were seen in single foetuses from different litters and their incidence was lower when compared with laboratory historical control data. The other alterations (anomalies and variations) recorded were noted both in control and treated groups, with a quite similar incidence.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant differences in AGDwere noted in male foetuses of mid- and high dose groups (approximately + 0.8% and + 3%, respectively) and in female foetuses of low and mid- dose groups (approximately -4% and -2.7%, respectively), compared to the control. Considering that the AGD reflects the prenatal androgenic exposure, with values physiologically higher in males than in females, the decrease observed in female foetuses represents a trend towards feminisation (reduction in AGD of females) and is not considered an adverse effect (i.e. an increase in AGD of females, suggesting masculinisation). The same applies to the increase observed in males, that also represents a trend towards masculinisation (increase in AGD of males) and is not considered an adverse effect (i.e. decrease in AGD of males, suggesting feminisation). Thus, these differences were considered incidental and non-adverse effects.
Details on embryotoxic / teratogenic effects:
The slight decrease noted in the mean foetal weight, for each sex as well as for both sexes combined, was not considered toxicologically relevant, considering the limited entity of the differences compared to control. The alterations noted at external, skeletal and visceral examinations of foetuses were con- sidered incidental and not treatment-related since they were quantitatively similar between control and treated groups with an absence of doses-related response. Based on these results, the NOAEL for developmental toxicity was considered to be 75 mg/kg bw/day.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 75 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: all parameters
Remarks on result:
other: effects were either toxicologically not relevant (decrease in foetal weight) or incidental and not treatment related (skeletal and visceral examinations)

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 15: Skeletal examination of foetuses

Organ

Malformation

Groups (mg/kg/day)

 

 

1 (0)

2 (18.75)

3 (37.5)

4 (75)

Pelvic girdle

Pubis not ossified

4(2)

3(2)

-

-

Ischium not ossified

-

1(1)

-

-

Skull

Presphenoid not ossified

2(2)

3(2)

1(1)

4(2)

Squamosal abnormal shape

-

-

-

1(1)

Cleft palate

-

1(1)

-

-

Forelimbs

Radius, ulna and humerus misshaped

-

-

1(1)

-

Ribs

Fused

-

-

2(1)

-

Thoracic vertebrae

Haemivertebra

-

-

1(1)

 

Absence of thoracic arch

-

-

1(1)

 

 

 

Foetus (litter)

Table 16: Visceralexaminationoffoetuses

Organ

Malformation

Groups (mg/kg/day)

 

 

1 (0)

2 (18.75)

3 (37.5)

4 (75)

Heart

Ventricle enlarged extreme

-

-

1(1)

-

Kidney/Ureter

Pelvic dilatation extreme/Enlarged extreme

-

-

-

1(1)

Foetus (Litter)

For other result tables, kindly refer to the attached background material section of the IUCLID.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the NOAEL for maternal and developmental toxicity was determined to be 75 mg/kg bw/day.
Executive summary:

A study was conducted to determine the developmental toxicity potential of C16 TMAC (purity: 98.5%), using prenatal developmental toxicity oral gavage study in female Sprague Dawley rats, according to the OECD Guideline 414, in compliance with GLP. All animals were administered the test substance during the gestation period, starting from Day 3 through Day 19 post coitum at the doses of 0, 18.75, 37.5, 75 mg/kg bw/day (25 animals in each group). Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post-mortem examination. Blood collection for haematology and hormone determination, in association with determination of the thyroid weight from all females was performed on Day 20 post coitum. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities. No animals died during the study. A total of three females were found not pregnant at necropsy: one in the control group, one in the low dose group (18.75 mg/kg bw/day) and one in the mid-dose group (37.5 mg/kg bw/day). One low dose female had unilateral implantation in the left horn and was not pregnant in the right one. The number of females with live foetuses on Day 20 post-coitum was: 24 in the control, low and mid-dose groups and 25 in the high dose group (75 mg/kg bw/day). Piloerection was the treatment-related clinical sign observed in all treated groups with a dose-related incidence. In Group 4 this sign was accompanied by hunched posture in few females. Minor signs, such as hair-loss and damaged ear were sporadically recorded during the study and were considered incidental. The slight statistically significant decreases in body weight observed in females of Group 4 was not considered of toxicological relevance due to the limited magnitude of the change. These changes were considered related to treatment, but not adverse. Statistically significant reduction in food consumption was observed in females of Group 4 starting from Day 6 post coitum through Day 15 post coitum. No differences were recorded on Day 18 post coitum and again a slight decrease was recorded on Day 20 post coitum. This change was considered not relevant because there was a recovery between the start and the end of treatment. The polychromasia observed in all treated groups was considered to be not adverse, even though the relation with the test substance cannot be excluded. Monocytosis observed in females dosed at 75 mg/kg bw/day was considered to be incidental. The increase of polychromatic cells in the peripheral blood smear even when the reticulocyte count is normal could suggests the premature release of erythroid cells from a marrow that is not hyperactive. Considering that: (a) no haematological findings were observed (e.g. anaemia) (b) no changes were recorded during the in-vivo phase or at post-mortem examination (c) no treatment-related effects were observed in previous studies (https://echa.europa.eu/it/registration-dossier/-/registered-dossier/14219/7/6/2). There were no indications of an effect of the test substance on the bone marrow or other tissues/organs which could led to an increase of the polychromatic cells, therefore this finding was considered to be not adverse. Due to the dose-relation observed between groups, the relation with the test substance cannot be definitively excluded. In thyroid hormone determination, no differences between control and treated females were recorded. There was no effect on the thyroid weight at any dose levels, compared to the control group. Statistically significant reductions in terminal body weight, gravid uterus weight and absolute weight gain (terminal body weight minus body weight at Day 0 post coitum minus gravid uterus), seen in females of Group 4, compared to controls were considered not adverse due to the limited magnitude of the change. No differences of toxicological relevance were noted in litter data and sex ratio. Statistically significant differences were noted in AGD of male foetuses of mid and high dose groups and in female foetuses of low and mid-dose groups, compared to the control. Considering that the AGD reflects the prenatal androgenic exposure, with values physiologically higher in males than in females, the increase observed in male foetuses represents a trend towards masculinisation (increase in AGD of males) and is not considered an adverse effect (i.e. decrease in AGD of males, suggesting feminisation). The same applies to the decrease observed in females, that also represents a trend towards feminisation (reduction in AGD of females) and is not considered an adverse effect (i.e. an increase in AGD of females, suggesting masculinisation). Thus, these differences were considered incidental and non-adverse effects. Furthermore, the AGD values were within the range of laboratory historical control data. At post-mortem, no treatment-related macroscopic changes were noted at low, medium and high dose. In microscopic examination, no treatment-related changes were noted in thyroid gland of females at low, medium and high dose. In external examination, small foetuses (<2.7 g) were present both in control and treated groups without a dose-relationship. One foetus in the mid-dose group had alterations classified as malformations: imperforate anus and absence of tail. These findings was considered unrelated to the test substance. In skeletal examination, malformations were detected in control and in treated groups without dose-relationship, in terms of foetuses and litters affected. The major and minor alterations detected at skeletal examination were considered incidental since they were quantitatively similar between groups or occurred in small foetuses (<2.7 g). In visceral examination, malformations were observed in one foetus of Group 3 and in one foetus of Group 4 (extremely enlarged ureter in association with kidney pelvic dilatation extreme). These findings were considered incidental since they were seen in single foetuses from different litters without a dose relationship, therefore a treatment-related effect is unlikely. The other alterations (anomalies and variations) recorded were noted both in control and treated groups, with a similar incidence. Overall, the study investigator concluded that the dosage of 75 mg/kg bw/day test substance induced slight maternal toxicity without adverse effects on pregnancy and developmental outcome. The dosages of 37.5 mg/kg bw/day and 18.75 mg/kg bw/day were well tolerated. The NOAEL for maternal toxicity could be considered at 75 mg/kg bw/day. No differences of toxicological relevance were observed between control and treated groups in the foetal development. The major and minor alterations noted at external, skeletal and visceral examinations of foetuses were considered incidental and not treatment-related since they were quantitatively similar between control and treated groups with an absence of dose-related response. Under the study conditions, the NOAEL for maternal and developmental toxicity was determined to be 75 mg/kg bw/day (Liberati, 2020).