1,2,4-Benzenetricarboxylic acid, tri-C9-11-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

- reproduction/developmental screening study (OECD 421): NOAEL >= 1000 mg/kg bw/day (m/f)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Jun - 23 Sep 2021

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:

adopted in 2016

Deviations:

yes

Remarks:

minor deviations related to weighing schedule, acclimatisation period length, and an extra vaginal smear from one animal (details were given under material and methods); no influence on study outcome

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Horst, Netherlands
- Females nulliparous and non-pregnant: yes
- Age at arrival: 7 - 8 weeks (males and females)
- Weight at arrival: 175 - 207 g (males); 144 - 190 g (females)
- Fasting period before study: no
- Housing: From arrival to pairing, animals were housed up to 5 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, Italy). Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed 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, Italy). Each cage tray held absorbent material which was inspected and changed daily. After mating, male were housed as before mating, whereas the females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5 × 26.6 × 18.5 cm).
- Diet: A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water: Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: An acclimatisation period of 32 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations. An acclimatisation period of 32 days was allowed before the start of treatment instead of approximately 3 weeks, as indicated in the Study Protocol.

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

IN-LIFE DATES: 04 Jun - 16 Aug 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The suspensions of the test item in corn oil was prepared using the following procedure:
1. the required amount of test item was weighed;
2. the required amount of vehicle was added;
3. the resulting mixture was left under magnetic stirring, at room temperature, for at least 30 min prior to dosing or analysis.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Concentration in vehicle: The preparations were made weekly at concentrations of 25, 75 and 250 mg/mL based on stability data. Concentrations were calculated and expressed in terms of test item as supplied.
- Amount of vehicle (if gavage): The test item was administered orally by gavage at a dose volume of 4 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1 (with the exception of one male of Group 1 (control) paired with two females, because of the death of one control male)
- Length of cohabitation: overnight or up to 14 days
- Positive identification of copulation: sperm identification, vaginal plug in situ or copulation plugs found in the cage tray
The female was paired with the same male until positive identification occurred or 14 days had elapsed.
- After successful mating each pregnant female was caged (how): individually

A parturition check was performed from Day 20 to Day 25 post coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was previously validated in the concentration range from 25 - 250 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85 - 115%; precision CV < 10%).
Samples of the formulations prepared in Weeks 1 and during the last week of treatment were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in the laboratory SOPs for suspensions (85 - 115% for concentration and CV < 10% for homogeneity).

Duration of treatment / exposure:

Males
Animals were dosed for 2 consecutive weeks prior to pairing, through the pairing period and thereafter until the day before necropsy, for a total of 32 days of treatment.

Females
Animals were dosed for 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 (for a total of 51 to 57 days of treatment).

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels have been selected in consultation with the Sponsor based on information from a preliminary non-GLP study.
- Fasting period before blood sampling for clinical biochemistry: no

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily (animals were checked for mortality early in each working day and also in the afternoon).
Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.
From the beginning of the study up to Day 3 of the pre-mating phase, three sessions of clinical observations were scheduled: 0.5 - 1 h post-dose, 2 - 2.5 h post-dose and 4 - 4.5 h post-dose. From Day 4 up to the end of the study, the animals were observed for clinical signs at approximately 2 - 2.5 h after the treatment.
All observations were recorded for individual animals.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed at allocation, then weekly from the start of dosing to termination.
Females were weighed at allocation, then weekly from the start of dosing 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 to necropsy.
- Animals were weighed on allocation and weekly from the start of treatment instead of weekly from allocation, as indicated in the Study Protocol.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from the beginning of treatment up to mating. Individual food consumption for mated 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 AND COMPOUND INTAKE: No

OTHER: The day before sacrifice, blood samples (1.1 mL) for thyroid hormone determination were collected from all parental males under isoflurane anaesthesia from the retro-orbital sinus.

Oestrous cyclicity (parental animals):

The assessment of oestrus cycles, by vaginal smears, was performed once daily in the morning in stock females and in allocated females for 2 weeks before the start of dosing.

From Day 1 of dosing up to positive identification of mating vaginal smears were taken in the morning to determine the following:
1. anomalies of the oestrus cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)

Vaginal smears were also taken from all females, before despatch to necropsy and the oestrus cycle phase recorded.
Due to an oversight, in one control female animal the vaginal smear was also taken on Day 12 of the post partum period (not only before the despatch to necropsy).
Data from vaginal smears performed on stock females were not tabulated in this report, but archived with all raw data.

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter; partial adjustment (e.g. 5 males and 3 females) was acceptable); excess pups were eliminated by random selection; no pups were eliminated when litter size dropped below the culling target of 8 pups/litter

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The males were killed on Day 19 of the mating phase after 32 days of treatment period.
- Maternal animals: The dams with live pups were killed on Day 14 post partum.

GROSS NECROPSY
- Gross necropsy consisted of external (surface and orifices) and internal examination.
- As a part of the necropsy procedure, blood samples (1.1 mL) for thyroid hormone determination were withdrawn from the abdominal vena cava of females under isoflurane anaesthesia.
All females were examined also for the following:
– number of visible implantation sites (pregnant animals)
– number of corpora lutea (pregnant animals)

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following tissues were prepared for microscopic examination: adrenal glands, clitoral gland, epididymides, kidneys, liver, mammary gland - females, mammary gland - males, ovaries with oviducts, seminal vesicles with coagulating glands, testes, thyroid, uterus - cervix, vagina; morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed
- The following tissues were weighed: adrenal glands, epididymides, kidneys, liver, ovaries with oviducts, prostate gland (dorsolateral and ventral), seminal vesicles with coagulating glands, spleen, testes, thymus (where present), thyroid, uterus - cervix

The examination was restricted to:
- all males and females in the control and high dose groups killed at term
- male of the control group sacrificed during the treatment period
- all abnormalities in all groups

Postmortem examinations (offspring):

SACRIFICE
- Culled pups were sacrificed on Day 4 post partum. All live pups were sacrificed on Day 14 post partum.

GROSS NECROPSY
- All pups found dead in the cage were examined for external and internal abnormalities.
- All culled pups sacrificed on Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection.
- All live pups sacrificed on Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
- No nipples/areolae from male pups were retained since no nipples were found at Day 13 post partum.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Thyroids were weighed from one male and one female pup selected for blood collection for hormone determination. For one dam, the thyroids were weighed from two different female pups because of the absence of male pups.

Statistics:

Group mean values, where possible, were calculated for all parameters. Data from females which did not mate, non-pregnant and with total litter loss were excluded from group mean.
Standard deviations were calculated as appropriate. For continuous variables, e.g. body weight, food consumption and organ weights, 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 histopathological findings was carried out by means of the nonparametric 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:

Males
Copulation index (%) = (no. of males with confirmed mating / no. of males cohabitated) x 100
Fertility index (%) = (no. of males which induced pregnancy / no. of males cohabitated) x 100

Females
Copulation index (%) = (no. of females with confirmed mating / no. of females cohabitated) x 100
Fertility index (%) = (no. of pregnant females / no. of females cohabitated) x 100

Males and females
Coital interval = The number of nights paired prior to the detection of mating
Gestation length = The time between the day of successful mating (Day post coitum) and the day of birth when parturition was defined complete (Day 0 post partum).

Offspring viability indices:

Pre-implantation loss (%) = ((no. of corpora lutea - no. of implantations) / no. of corpora lutea) x 100
Pre-natal loss (%) = ((no. of visible implantations - live litter size at birth) / no. of visible implantations) x 100
Pup loss at Day 0 post partum (%) = ((total litter size - live litter size) / total litter size) x 100
Postnatal loss on Day 4 post partum (before culling) (%) = ((live litter size at birth - live litter size at Day 4, before culling) / live litter size at birth) x 100
Postnatal loss on Day 13 post partum (after culling) (%) = ((live litter size on Day 4, after culling - live litter size on Day 13) / live litter size on Day 4, after culling) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

Males
No clinical signs were observed in all treated males before pairing and during the mating phase.

Females
No clinical signs were observed in all treated females before and during mating, during the gestation period and throughout the post partum phase.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One case of unscheduled death occurred in the control group: one male animal was sacrificed for humane reasons on Day 7 of the pre-mating phase.
At post mortem examination, the gavage catheter or cannula was present in the oesophagus. Error at gavage procedure was considered to represent the cause of death.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight of treated and control animals was comparable throughout the entire study.
Mean body weight gain of mid-dose males was found statistically significantly lower than controls on Day 15 of the mating phase. A statistically significant decrease in body weight gain was also observed for females of the low and mid-dose groups on Day 8 of the premating phase. Being isolated events and without a dose-related trend, these cases were not considered to be treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were seen in food consumption during the whole duration of the study.

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):

Thyroid hormones
No changes were recorded in thyroid hormones of parental male and female animals at the end of the treatment period.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study:
- thyroid hormones
- accessory sex organ weight and histopathology
- anogenital distance
- coagulating gland weight
- sex organ weight and histopathology
- oestrus cyclicity
- genital abnormalities
- nipple development
- thyroid weight and histopathology
- vaginal smears
- fertility
- fetal development
- gestation length
- litter size
- litter viability
- litter / pup weight
- number of implantations, corpora lutea
- number of live births
- pre-implantation / pre-natal loss
- reproduction
- sex ratio

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted in the sex organs/tissues of males and females at 1000 mg/kg bw/day, when compared to controls.
There were no treatment-related microscopic observations in the testes examined with PAS special stain.
The sporadic microscopic observations in control and/or treated males were consistent with those known to occur spontaneously in untreated SD rats of the same age and were considered incidental and unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The oestrus cyclicity of the treated females monitored during the pre-mating period, for a total of 14 days, was not affected by treatment. The mean number of oestrus cycles observed in treated animals and controls was 3 cycles.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Three mid-dose females and two high dose females were sacrificed on Days 26 and 27 post coitum and found not pregnant.
One high dose female showed total litter loss on Day 3 post partum and therefore was sacrificed on Day 4 post partum.
Two control females did not show evidence of copulation after 2 weeks of mating. One of the two gave birth at the end of the gestation phase. One female was sacrificed on Day 43 of the mating phase and found not pregnant.
The number of dams with live pups on Day 14 post partum was: 9 in the control, 10 in the low dose (100 mg/kg/day), 7 in mid (300 mg/kg/day) and high dose (1000 mg/kg/day) groups. See 'Any other information on results incl. tables', Table 1.

The number of copulatory plugs (mean value) found in the cage was 3 each in control and in the treated groups.
Copulatory and fertility indices were considered to be unaffected by the administration of the test item.

Males and females
The copulatory indices were 90% for controls and 100% in all treated groups of both sexes.
The fertility indices were 88.9% in males and 90% in females for controls and for both sexes, 100% in low dose animals, 70 and 80% in mid and high dose animals, respectively.
Animals, both in control and treated groups, mated after 2/3 days (mean pre-coital interval) of cohabitation.

The number of implantations, live litter size, pre-implantation and pre-natal loss did not show treatment-related differences. Mean gestation period was comparable between control and treated animals. All dams give birth between Days 22 and 23 post coitum.

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Some clinical signs, such as small size, coldness to touch, apparently no food intake and difficulty in breathing, were observed in some pups of treated groups. These findings were mostly noted in litters from individual dam (2 dams in Group 2, 1 dam in Group 3, and 2 dams in Group 4). Most of pups did not recover from these signs and were found dead or missing.
In addition, an increase incidence of found dead pups was recorded in treated groups. This trend correlates with the slight increase in pup loss percentage observed in the high dose group.
Since clinical signs, losses and/or missing pups were mainly noted in individual animals, the correlation to treatment was not clear. See 'Any other information on results incl. tables', Table 2 and 3.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Total litter size and live litter size at birth and on Days 1, 4 and 13 post partum were comparable between groups. The value of post-natal loss was observed to be greater for the high dose group on Day 4 post partum, but without statistical significance. This occurrence was mainly due to one dam which lost its litter on Day 3 post partum.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences in litter weight and mean pup weight were observed among treated and control animal, from birth up to Day 13 post partum.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid hormones
Pups – Day 14 post partum
No changes were recorded in thyroid hormones of male and female pups.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance was comparable between control and treated groups.

Nipple retention in male pups:

not examined

Description (incidence and severity):

Not examined as no nipples were found on Day 13 post partum.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

No findings were observed in decedent pups, nor in pups sacrificed on Days 4 and 14 post partum.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Sex ratios at birth, on Days 4 and 14 post partum showed a decrease in male pups percentage of the high dose group, compared to controls, without statistical significance. This difference was not considered to be treatment-related.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Critical effects observed:

no

Reproductive effects observed:

no

Table 1: Summary of reproduction data – Group data

Observations	Dose levels (mg/kg bw/day)
	0	100	300	1000
No. Dams with positive identification of mating	9	10	10	10
Oestrus cycle (mean number)	3	3	3	3
No. Dams That Conceived	9	10	7	8
Percent Dams Conceived/total	90.0	100.0	70.0	80.0
Positive identification of mating (1-5 days)	8	10	10	9
Positive identification of mating (6-14 days)	0	0	0	1
No. Dams Died During Study	0	0	0	0
No. Dams with Resorptions only	0	0	0	0
No. of Litters	9	10	7	8
Total No. Corpora Lutea	135	145	99	115
Mean No. Corpora Lutea/Pregnancy	15.0	14.5	14.1	14.4
Total No. Implantation	135	145	99	115
Mean No. Implants/Pregnancy	15.0	14.5	14.1	14.4
Total No. Resorptions	0	0	0	0
Preimplantation Loss (%)	0.0	0.0	0.0	0.0
Pre natal Loss (%)	24.38	18.99	10.36	23.56

 

Table 2: Litter data at birth on Day 1 and on Day 4 post partum (before culling) of females with live pups at Day 1 post partum – Group mean data

Group		At birth			On Day 1 post partum			On Day 4 post partum
		Total litter size	Live litter size	Post natal loss (%)	Live litter size	Litter weight (g)	Mean pup weight (g)	Live litter size	Post natal loss (%)	Litter weight (g)	Mean pup weight (g)
1	Mean	11.67	11.56	0.86	11.33	78.72	7.07	11.33	1.62	114.89	10.53
	SD	4.18	4.16	2.57	4.03	24.95	0.69	4.03	3.26	32.74	1.46
	N	9	9	9	9	9	9	9	9	9	9
2	Mean	13.00	11.80	8.92	11.50	72.18	6.37	11.30	4.47	103.17	9.33
	SD	2.21	2.53	12.69	2.80	14.54	0.60	2.67	6.47	16.80	1.32
	N	10	10	10	10	10	10	10	10	10	10
3	Mean	13.43	12.71	5.09	12.29	81.54	6.70	12.29	3.33	118.01	9.77
	SD	2.64	2.69	9.21	2.69	13.05	0.59	2.69	5.76	16.82	1.21
	N	7	7	7	7	7	7	7	7	7	7
4	Mean	13.00	11.25	13.25	10.50	70.23	6.59	10.25	17.86	106.26	9.43
	SD	3.55	4.80	25.27	5.83	39.27	1.20	6.27	36.43	40.60	1.96
	N	8	8	8	8	8	8	8	8	7	7
Statistical analysis: 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 3: Clinical signs of pups – Group incidence

Groups	1		2		3		4
Litters observed	9		10		7		8
	Affected no.	%	Affected no.	%	Affected no.	%	Affected no.	%
Small/Smaller than others	2	22.2	3	30.0	1	14.3	2	25.0
Cold/Cold to touch	0	0.0	1	10.0	1	14.3	3	37.5
Apparently no food intake	0	0.0	2	20.0	0	0.0	3	37.5
Difficulty in breathing	0	0.0	0	0.0	1	14.3	0	0.0
Pale	0	0.0	1	10.0	0	0.0	0	0.0

 

Conclusions:

The test item had no effect on reproductive performance. The NOAEL was concluded to be >/= 1000 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Prenatal developmental toxicity test

The developmental toxicity of the registered substance was investigated in a study performed according to OECD guideline 414 under GLP conditions (Croda Europe Limited, 2022). The registered substance was administered once daily via gavage to groups of 25 mated Sprague-Dawley female rats at doses of 100, 300, and 1000 mg/kg bw in corn oil from gestation Day 3 up to Day 19. The dose volume was set at 4 mL/kg bw.

No treatment-related clinical signs were observed in dams. In addition, the test item did not cause changes in body weight. A slight decrease in the body weight gain and food consumption was noted in single instances at 300 and/or 1000 mg/kg bw/day when compared to controls and these occurrences were not considered adverse. No treatment-related findings were noted at necropsy of females or at thyroid weight (absolute or relative to brain weight). TSH values were found statistically significantly higher in females at 1000 mg/kg bw/day, but no changes were observed for T3 and T4 values. Microscopic analysis of thyroid did not reveal findings at any dose level. Thus, in the absence of other related findings (i.e. T3, T4, thyroid weight and/or histopathological changes), the increase of TSH values were considered to be unrelated to treatment.

The number of corpora lutea, implantations, viable foetuses, implantation loss, intrauterine deaths and sex ratios were comparable between control and treated groups. No treatment related effects were seen in the mean litter and foetal weight. The external examination of foetuses, including the measurement of the anogenital distance, did not show differences in treated animals at all dose levels, when compared to the control group. No treatment-related findings were noted at skeletal and visceral examinations. Indeed, no evidence of teratogenicity was noted up to and including the dose level of 1000 mg/kg bw/day.

Based on the available data, no changes that could be considered adverse were observed at any of the dose level investigated. Therefore, the No Observed Adverse Effect Level (NOAEL) for general toxicity and developmental toxicity was concluded to be 1000 mg/kg bw/day.

 

Reproductive/Developmental screening test

The reproductive/developmental toxicity of the registered substance was investigated in a study performed according to OECD guideline 421 under GLP conditions (Croda Europe Limited, 2023). The registered substance was administered once daily via gavage to groups of 10 Sprague-Dawley rats per sex at doses of 100, 300, and 1000 mg/kg bw in corn oil. A control group received the vehicle alone. The dose volume was 4 mL/kg bw. Males were treated for 14 days prior to pairing, and during pairing with females until the day before necropsy up to a total of 32 days. Females were treated for 14 days prior to pairing, during pairing, and throughout the gestation and lactation periods until Day 13 post partum, up to a total of 51 to 57 days.

Litter data (litter size, pup loss, litter weights and mean pup weights), anogenital distance, thyroid hormone levels, and thyroid weights in pups at Day 14 post partum were unaffected by treatment. No toxicological significance was attributed to the slight reduction in male percentage observed in the high dose animals or to the slight increase in mortality and clinical signs of some litters from high dose dams. Some clinical signs, such as small size, coldness to touch, apparently no food intake and difficult in breathing, were observed in some pups from few treated dams of each treated group. In addition, and as compared to the control group, an increased incidence of pups found dead was recorded in all treated groups. Since clinical signs, losses and/or missing pups were mainly noted in individual animals, a clear correlation to treatment could not be drawn. No necropsy findings were observed in decedent pups, nor in pups sacrificed on Days 4 and 14 post partum. No nipples were observed in male pups.

Based on the available data, no changes that could be considered adverse were observed at any of the dose level investigated. Therefore, the No Observed Adverse Effect Level (NOAEL) for developmental toxicity was concluded to be 1000 mg/kg bw/day, for both male and female pups (for detailed information on systemic and reproduction toxicity please refer to the fertility section above).

 

Conclusion for developmental toxicity
Developmental toxicity was investigated in a prenatal developmental toxicity test performed according to OECD guideline 414 under GLP conditions and a reproductive/developmental screening test performed according to OECD guideline 421 under GLP conditions. No adverse effects to the parental animals, fertility, or the pups were observed up to the highest dose and a NOAEL of 1000 mg/kg bw/day was concluded in both studies.

Effects on developmental toxicity

Description of key information

- reproduction/developmental screening study (OECD 421): NOAEL >= 1000 mg/kg bw/day (m/f)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jul - 07 Oct 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

minor deviations related to source of animals, shipping logistics, freezer warming after all analyses were performed, and purity of ethanol fixation (details were given under material and methods); no influence on study outcome

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The animals were ordered from Envigo RMS Srl, San Pietro al Natisone (UD), Italy instead of Charles River Italia S.p.A., Calco (Lecco, Italy).
- Age at animal order: 9 week females; 11 week males
- Weight at animal order: 175 - 225 g females; at least 340 g males
- Fasting period before study: no
- Housing: no more than 5 per cage in clear polysulfone cages before mating and after mating
- Diet: A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water: water bottles, ad libitum
- Acclimation period: An acclimatisation period of approximately 2 weeks was allowed before the start of mating.

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

IN-LIFE DATES: 15 Jul - 15 Aug 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was suspended in the vehicle to reach the required concentrations of 25, 75 and 250 mg/mL. The preparations were made up to weekly interval based on the stability data. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Amount of vehicle (if gavage): The test item was administered orally by gavage at a dose volume of 4 mL/kg bw. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated previously in the range from 25 - 250 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85 - 115%; precision CV < 10%). A 28 h stability at room temperature and a 10 d stability at room temperature were verified in the range from 25 - 250 mg/mL.
Samples of the formulations prepared in Week 1 and in the last week were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in the laboratory SOPs for suspensions (85 - 115% for concentration and CV% < 10% for
homogeneity).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 / 1
- Length of cohabitation: Overnight (until a positive identification of mating was made)
- Proof of pregnancy: 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

Duration of treatment / exposure:

All animals were dosed once a day from Day 3 through Day 19 post coitum.

Frequency of treatment:

daily, 7 days/week

Duration of test:

Day 20 post coitum

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels have been selected by the Sponsor based on previous non-GLP preliminary study
- Fasting period before blood sampling for (rat) dam thyroid hormones: not specified
- Time of day for (rat) dam blood sampling: on Day 20 post coitum

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily (animals were checked for mortality early in each working day and also in the afternoon)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum

FOOD CONSUMPTION: Yes
Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 20 post coitum
- Organs examined: Uterus and ovaries. From all females the thyroid and the brain were weighed, fixed and preserved in 10% neutral buffered formalin. The thyroid weight was determined after fixation. The ratios of organ weight to brain weight was calculated for each animal. The thyroid was also examined histopathologically.
All animals were killed by carbon dioxide inhalation on Day 20 post coitum and necropsied.

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; number and sex of dead foetuses (foetuses at term without spontaneous movements); number of intra-uterine deaths; gross evaluation of placentae

Blood sampling:

- Serum: Yes
- Plasma: no
- Volume collected: Approximately 1 mL
- The Principal Investigator was inadvertently not notified by email of the shipping date of the serum samples.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter (soft tissue and skeletal examination, respectively)
- Anogenital distance of all live rodent pups: yes
- Foetuses for skeletal examination were fixed in 99% ethanol instead of 95%, as stated in the Study Protocol.

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. The criterion for statistical significance was p < 0.05.

Indices:

Pre-implantation loss was calculated as a percentage from the formula:
((no. of corpora lutea - no. of implantations) x 100) / no. of corpora lutea

Post-implantation loss was calculated as a percentage from the formula:
((no. of implantations - no. of live foetuses) x 100) / no. of implantations

Total implantation loss was calculated as a percentage from the formula:
((no. of corpora lutea - no. of live foetuses) x 100) / no. of corpora lutea

Sex ratios of the foetuses were calculated as the percentage of males.
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:

Historical control data for T3, T4, and TSH were provided.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Sign of hairloss was observed in one single animal of the low dose group, from Day 10 to 19 of the gestation phase. This finding was associated with a slight decrease in the body weight gain on Day 12 post coitum, fully recovered thereafter.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significantly decrease of the body weight gain was recorded on gestation Day 12 in the high dose group (-26%), when compared to controls. The animals then recovered, therefore this reduction was not considered adverse. No other differences occurred during gestation.
Body weight at termination and the absolute weight gain of females were unaffected by treatment with the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A slight but statistically significant decrease in food consumption was observed in the mid and high dose groups on gestation Day 6 (up to -13% compared to the control group). This effect was transient occurrence and was not considered adverse. No other changes were seen between treated and control groups.

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:

effects observed, non-treatment-related

Description (incidence and severity):

TSH was statistically higher than controls in females dosed at 1000 mg/kg bw/day (+32%). Even though a number of these animals showed values slightly outside the range of historical data (12 out of 25), no other related changes were observed (i.e. T3, T4, thyroid weight and/or histopathological changes); the above finding was therefore considered to be unrelated to treatment (see 'Any other information on results incl. tables', Table 1 (results) and Table 2 (historical control values)).

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study:
- thyroid hormones
- anogenital distance
- genital abnormalities
- thyroid weight and histopathology
- gravid uterus weight
- litter size
- litter / pup weight
- number of implantations, corpora lutea
- number of embryonic fetal deaths and viable fetuses
- post- / pre-implantation loss
- presence of anomalies (external, visceral, skeletal)
- sex ratio

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Gravid uterus weight was unaffected by treatment with the test item.
There were no treatment-related thyroid gland weight changes (absolute and relative to brain weights) in treated females, when compared to controls.
Any organ weight variations were within the range of expected variations in SD rats of the same age and considered incidental and unrelated to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic observations in treated females receiving the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic observations in the thyroid gland of females
receiving the test item.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no differences in the number of implantation loss and intrauterine deaths between control and treated groups.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no differences in total litter losses by resorption between control and treated groups.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no differences in the number early or late resorptions between control and treated groups.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no differences in the number of dead fetuses between control and treated groups.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were no differences in the number of implantations between control and treated groups.

Other effects:

not examined

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effect observed up to and including the highest dose tested.

Dose descriptor:

NOAEL

Remarks:

maternal general toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effect observed up to and including the highest dose tested.

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean litter and foetal weights (both sexes) did not differ between control and treated groups.
Isolated cases of small foetuses (body weight below 2.7 g) were found in control, low and mid-dose groups with comparable incidence.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The number of live foetuses did not differ between control and treated groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The percentage of males respect to females did not differ between control and treated groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean litter size and weights did not differ between control and treated groups.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

No differences in the anogenital distance were seen in either male or females foetuses between all treated and control groups.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Abnormal shape of the hindlimb was observed in two foetuses from litters receiving the vehicle. In addition, one of those two foetuses showed bent tail.
No other external findings were recorded in all groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No major abnormalities were found. Minor abnormalities or variations occurred in all groups and included for example altered ossification (asymmetrical, incomplete or no ossification) of several bones of the skull, sternebrae, forepaws, thoracic vertebrae, pelvic girdle and short or rudimentary supernumerary ribs (14th). The incidence of the affected litters in treated groups was similar or even lower than observed in the control group or without dose relation. Therefore, these findings were considered unrelated to treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No major abnormalities were found. The incidences of foetuses or litters with anomalies or variations did not suggest any test item effect.

Other effects:

not examined

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed up to and including the highest dose tested

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: skull

skeletal: forelimb

skeletal: sternum

skeletal: supernumerary rib

skeletal: vertebra

skeletal: pelvic girdle

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

visceral/soft tissue: central nervous system

visceral/soft tissue: male reproductive system

Description (incidence and severity):

The observed findings occurred as isolated or incidental findings without a dose-response relationship or were comparable to the control group. Therefore, they were considered non-treatment related.

Developmental effects observed:

no

Table 1: Thyroid hormone determination on Day 20 post coitum – Group mean data

Parameter/units		Group
		1	2	3	4
T3 nmol/L	N	25	25	25	25
	Mean	0.963	0.981	1.000	0.975
	SD	0.126	0.070	0.185	0.137
T4 nmol/L	N	25	25	25	25
	Mean	24.3	23.0	24.1	23.0
	SD	5.8	6.5	7.4	6.4
TSH ng/mL	N	25	25	25	25
	Mean	8.50	10.49	10.46	11.22+
	SD	2.17	3.49	3.19	3.39
* = 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 N = number per group SD = standard deviation

 

Table 2: Historical data for Sprague Dawley rat

	Males	Females
T3 historical data for Sprague Dawley rat in serum with the kit Beckman Coulter – Immunotech s.r.o., reference IM1699
Mean	0.82	0.86
SD	0.22	0.25
Minimum	0.40	0.39
Maximum	1.57	1.61
Median	0.78	0.79
5e Centile	0.54	0.55
95e Centile	1.30	1.35
Number	109	251
Years of reference	2020 - 2021	2020 - 2021
Number of studies considered	10	14
T4 historical data for Sprague Dawley rat in serum with the kit Beckman Coulter – Immunotech s.r.o., reference IM1447
Mean	45	22
SD	7.45	7.63
Minimum	29	13
Maximum	68	54
Median	44	19
5e Centile	35	16
95e Centile	57	38
Number	150	252
Years of reference	2020 - 2021	2020 - 2021
Number of studies considered	14	14
TSH historical data for Sprague Dawley rat in serum with the kit Institute of Isotopes Ltd., reference RK-554
Mean	10.3	7.2
SD	5.45	2.56
Minimum	1.6	2.5
Maximum	43.6	18.9
Median	8.9	6.7
5e Centile	5.1	3.8
95e Centile	18.3	11.2
Number	150	252
Years of reference	2020 - 2021	2020 - 2021
Number of studies considered	14	14

Conclusions:

Under the present test conditions, the test item had no effect on intrauterine development. The NOAEL was concluded to be >/= 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Prenatal developmental toxicity test

The developmental toxicity of the registered substance was investigated in a study performed according to OECD guideline 414 under GLP conditions (Croda Europe Limited, 2022). The registered substance was administered once daily via gavage to groups of 25 mated Sprague-Dawley female rats at doses of 100, 300, and 1000 mg/kg bw in corn oil from gestation Day 3 up to Day 19. The dose volume was set at 4 mL/kg bw.

No treatment-related clinical signs were observed in dams. In addition, the test item did not cause changes in body weight. A slight decrease in the body weight gain and food consumption was noted in single instances at 300 and/or 1000 mg/kg bw/day when compared to controls and these occurrences were not considered adverse. No treatment-related findings were noted at necropsy of females or at thyroid weight (absolute or relative to brain weight). TSH values were found statistically significantly higher in females at 1000 mg/kg bw/day, but no changes were observed for T3 and T4 values. Microscopic analysis of thyroid did not reveal findings at any dose level. Thus, in the absence of other related findings (i.e. T3, T4, thyroid weight and/or histopathological changes), the increase of TSH values were considered to be unrelated to treatment.

The number of corpora lutea, implantations, viable foetuses, implantation loss, intrauterine deaths and sex ratios were comparable between control and treated groups. No treatment related effects were seen in the mean litter and foetal weight. The external examination of foetuses, including the measurement of the anogenital distance, did not show differences in treated animals at all dose levels, when compared to the control group. No treatment-related findings were noted at skeletal and visceral examinations. Indeed, no evidence of teratogenicity was noted up to and including the dose level of 1000 mg/kg bw/day.

Based on the available data, no changes that could be considered adverse were observed at any of the dose level investigated. Therefore, the No Observed Adverse Effect Level (NOAEL) for general toxicity and developmental toxicity was concluded to be 1000 mg/kg bw/day.

 

Reproductive/Developmental screening test

The reproductive/developmental toxicity of the registered substance was investigated in a study performed according to OECD guideline 421 under GLP conditions (Croda Europe Limited, 2023). The registered substance was administered once daily via gavage to groups of 10 Sprague-Dawley rats per sex at doses of 100, 300, and 1000 mg/kg bw in corn oil. A control group received the vehicle alone. The dose volume was 4 mL/kg bw. Males were treated for 14 days prior to pairing, and during pairing with females until the day before necropsy up to a total of 32 days. Females were treated for 14 days prior to pairing, during pairing, and throughout the gestation and lactation periods until Day 13 post partum, up to a total of 51 to 57 days.

Litter data (litter size, pup loss, litter weights and mean pup weights), anogenital distance, thyroid hormone levels, and thyroid weights in pups at Day 14 post partum were unaffected by treatment. No toxicological significance was attributed to the slight reduction in male percentage observed in the high dose animals or to the slight increase in mortality and clinical signs of some litters from high dose dams. Some clinical signs, such as small size, coldness to touch, apparently no food intake and difficult in breathing, were observed in some pups from few treated dams of each treated group. In addition, and as compared to the control group, an increased incidence of pups found dead was recorded in all treated groups. Since clinical signs, losses and/or missing pups were mainly noted in individual animals, a clear correlation to treatment could not be drawn. No necropsy findings were observed in decedent pups, nor in pups sacrificed on Days 4 and 14 post partum. No nipples were observed in male pups.

Based on the available data, no changes that could be considered adverse were observed at any of the dose level investigated. Therefore, the No Observed Adverse Effect Level (NOAEL) for developmental toxicity was concluded to be 1000 mg/kg bw/day, for both male and female pups (for detailed information on systemic and reproduction toxicity please refer to the fertility section above).

 

Conclusion for developmental toxicity
Developmental toxicity was investigated in a prenatal developmental toxicity test performed according to OECD guideline 414 under GLP conditions and a reproductive/developmental screening test performed according to OECD guideline 421 under GLP conditions. No adverse effects to the parental animals, fertility, or the pups were observed up to the highest dose and a NOAEL of 1000 mg/kg bw/day was concluded in both studies.

Toxicity to reproduction: other studies

Description of key information

- reproduction/developmental screening study (OECD 421): NOAEL >= 1000 mg/kg bw/day (m/f)

Additional information

Prenatal developmental toxicity test

The developmental toxicity of the registered substance was investigated in a study performed according to OECD guideline 414 under GLP conditions (Croda Europe Limited, 2022). The registered substance was administered once daily via gavage to groups of 25 mated Sprague-Dawley female rats at doses of 100, 300, and 1000 mg/kg bw in corn oil from gestation Day 3 up to Day 19. The dose volume was set at 4 mL/kg bw.

No treatment-related clinical signs were observed in dams. In addition, the test item did not cause changes in body weight. A slight decrease in the body weight gain and food consumption was noted in single instances at 300 and/or 1000 mg/kg bw/day when compared to controls and these occurrences were not considered adverse. No treatment-related findings were noted at necropsy of females or at thyroid weight (absolute or relative to brain weight). TSH values were found statistically significantly higher in females at 1000 mg/kg bw/day, but no changes were observed for T3 and T4 values. Microscopic analysis of thyroid did not reveal findings at any dose level. Thus, in the absence of other related findings (i.e. T3, T4, thyroid weight and/or histopathological changes), the increase of TSH values were considered to be unrelated to treatment.

The number of corpora lutea, implantations, viable foetuses, implantation loss, intrauterine deaths and sex ratios were comparable between control and treated groups. No treatment related effects were seen in the mean litter and foetal weight. The external examination of foetuses, including the measurement of the anogenital distance, did not show differences in treated animals at all dose levels, when compared to the control group. No treatment-related findings were noted at skeletal and visceral examinations. Indeed, no evidence of teratogenicity was noted up to and including the dose level of 1000 mg/kg bw/day.

Based on the available data, no changes that could be considered adverse were observed at any of the dose level investigated. Therefore, the No Observed Adverse Effect Level (NOAEL) for general toxicity and developmental toxicity was concluded to be 1000 mg/kg bw/day.

 

Reproductive/Developmental screening test

The reproductive/developmental toxicity of the registered substance was investigated in a study performed according to OECD guideline 421 under GLP conditions (Croda Europe Limited, 2023). The registered substance was administered once daily via gavage to groups of 10 Sprague-Dawley rats per sex at doses of 100, 300, and 1000 mg/kg bw in corn oil. A control group received the vehicle alone. The dose volume was 4 mL/kg bw. Males were treated for 14 days prior to pairing, and during pairing with females until the day before necropsy up to a total of 32 days. Females were treated for 14 days prior to pairing, during pairing, and throughout the gestation and lactation periods until Day 13 post partum, up to a total of 51 to 57 days.

Litter data (litter size, pup loss, litter weights and mean pup weights), anogenital distance, thyroid hormone levels, and thyroid weights in pups at Day 14 post partum were unaffected by treatment. No toxicological significance was attributed to the slight reduction in male percentage observed in the high dose animals or to the slight increase in mortality and clinical signs of some litters from high dose dams. Some clinical signs, such as small size, coldness to touch, apparently no food intake and difficult in breathing, were observed in some pups from few treated dams of each treated group. In addition, and as compared to the control group, an increased incidence of pups found dead was recorded in all treated groups. Since clinical signs, losses and/or missing pups were mainly noted in individual animals, a clear correlation to treatment could not be drawn. No necropsy findings were observed in decedent pups, nor in pups sacrificed on Days 4 and 14 post partum. No nipples were observed in male pups.

Based on the available data, no changes that could be considered adverse were observed at any of the dose level investigated. Therefore, the No Observed Adverse Effect Level (NOAEL) for developmental toxicity was concluded to be 1000 mg/kg bw/day, for both male and female pups (for detailed information on systemic and reproduction toxicity please refer to the fertility section above).

 

Conclusion for developmental toxicity
Developmental toxicity was investigated in a prenatal developmental toxicity test performed according to OECD guideline 414 under GLP conditions and a reproductive/developmental screening test performed according to OECD guideline 421 under GLP conditions. No adverse effects to the parental animals, fertility, or the pups were observed up to the highest dose and a NOAEL of 1000 mg/kg bw/day was concluded in both studies.

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008 the data on reproductive toxicity are conclusive but not sufficient for classification.