Fatty acids, C10-12, esters with polylactic acid, sodium salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Screening test, oral (OECD 421): NOAEL (systemic, rat, m/f) = 750 mg/kg bw/day; NOAEL (reproduction) = 750 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Oct 2020 - 10 May 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 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Sprague Dawley SD

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice as it is generally accepted by regulatory authorities and there are ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italy S.p.A., Calco (Lecco), Italy
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: Males: 197 - 250 g; Females: 186 - 211 g
- Housing:
Arrival to mating: clear polysulfone solid bottomed cages, up to 5 per sex.
Mating: clear polysulfone cages with a stainless steel mesh lid and floor, one male and one female per cage.
After mating: males were housed as they were before mating, females were individually housed in solid bottomed cages for the gestation period, birth and lactation.
- Diet: 4 RF 21 (supplier: Mucedola S.r.l., Settimo Milanese (MI), Italy), ad libitum
- Water: In water bottles, ad libitum
- Acclimation period: 27 days

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

IN-LIFE DATES: From: 14 Oct 2020 To: 14 Dec 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing formulations were prepared weekly by suspending the appropriate amount of the test material in the vehicle (corn oil) at 40 °C under magnetic stirring for at least 16 h yielding the final concentrations of 62.5, 125 and 187.5 mg/mL. Each day, approx. 16 h prior to administration, the dosing formulations were again heated to 40 °C under magnetic stirring.

VEHICLE
- Justification for use and choice of vehicle: In a preliminary non-GLP toxicity test (ERBC study no. E0475), irritating effects in the stomach of rats were observed. Corn oil is known to alleviate local irritating effects.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: Sperm identification, vaginal plug in situ or copulation plugs found in the cage tray, referred to as Day 0 post coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In ERBC Study no. A3808, a 20 hour stability at 40°C and an 8 day stability at room temperature were verified in the range from 10 to 250 mg/mL. In ERBC Study no. A3991, stability at 40°C was extended up to 24 hours in the range from 62.5 to 187.5 mg/mL. Final results for all concentration levels were within the acceptability limits for concentration (80 - 120%) and homogeneity (Coefficient of Variation, CV < 10%). Samples of the dosing formulations prepared on Week 1 and Week 5 were analysed to verify the homogeneity and concentration. Results of the analyses were within the acceptability limits for suspensions as given above.

Duration of treatment / exposure:

(P) Males: 34 to 35 days (14 days pre-maiting and during pairing with females until the day before necropsy)
(P) Females: 42 to 55 days (14 days pre-mating until post partum Day 13)

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on information from a preliminary non-GLP compliant study (ERBC Study no.: E0475). In this study, a 2-week preliminary oral toxicity study in rats, carried out at dose levels of 100, 300 and 1000 mg/kg bw/day, 2 out of 4 male and 1 out of 4 female animals dosed at 1000 mg/kg bw/day died following dosing. Clinical signs observed in the majority of animals dosed at 1000 mg/kg bw/day included hunched posture, swollen abdomen, rales, dyspnoea, pallor and decreased activity. Distended gastrointestinal tract with gas (stomach and/or jejunum and/or ileum and/or caecum and/or colon) was observed in some animals dosed at 1000 mg/kg bw/day, including the deceased animals. No further significant signs of toxicity were observed in these animals. In a subsequent 4 week toxicity study in rats (ERBC No. A3991), performed at 100, 300 and 750 mg/kg bw/day, 1 high dose male died prematurely. Salivation was observed after dosing in treated animals, with a dose-related trend. Abnormal size of the stomach was observed at post mortem macroscopic observations. No clinical signs or other signs of systemic toxicity were seen in animals dosed at 300 and 100 mg/kg bw/day in both studies. Therefore, in this study the NOAEL was considered to be 750 mg/kg bw/day.
The effects observed at 750 and 1000 mg/kg bw/day (including mortalities) were the result of local irritation of the test item to the stomach. On the basis of these results, as no other systemic effects were observed at 750 mg/kg bw/day, this dose level was considered to be a suitable high dose level in this study.

- Fasting period before blood sampling for clinical biochemistry: No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once daily during the study, each animal was observed for clinical signs. Animals were checked twice daily for mortality.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly. Females were weighed weekly until positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were weighed on Days 1, 4, 7 and 13 post partum and on the day of necropsy.

FOOD CONSUMPTION: Yes
- The weight of food consumed by each cage of males and females were recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

WATER CONSUMPTION: No

OTHER:
Thyroid hormone determination (T3, T4 and TSH): Blood collection was performed only for animals at termination (males - under isoflurane anaesthesia from the sublingual vein, females - under isoflurane anaesthesia from the abdominal vena cava). No samples were taken from animals sacrificed for humane reasons.

Oestrous cyclicity (parental animals):

Before allocation - Stock females:
Oestrous cycle was monitored by vaginal smears in all females for 2 weeks before allocation into study groups, in order to exclude from the study females with irregular cycles.

Females allocated to groups:
Vaginal smears were taken in the morning starting from Day 1 of dosing up to positive identification of copulation. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous 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 oestrous cycle phase recorded. No vaginal smears were taken from females sacrificed for humane reasons.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Testis weight and epididymis weight. A detailed qualitative evaluation of testes was performed on all control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 post partum: yes
- A maximum of 8 pups/litter (4/sex/litter as closely as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, sex confirmation by gonads inspection.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities.

OTHER:
Thyroid hormone determination (T3, T4 and TSH): On Days 4 and 14 post partum, as part of the necropsy procedure, blood samples (approximately 1.0 mL each aliquot) were withdrawn from at least one culled male and one culled female from each litter. Samples from different pups per sex were pooled if necessary. Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture).

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were killed after the mating of all females or after 34 or 35 days of treatment.
- Maternal animals: The females with live pups were killed on Day 14 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. The females which did not give birth 25 days after positive identification of mating were killed shortly after.

GROSS NECROPSY
- Gross necropsy consisted of examination of the external surface and orifices.
- All females were examined for the number of visible implantation sites (pregnant animals). Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following tissues were weighed and prepared for microscopic examination:
Adrenal glands, Brain (cerebrum, cerebellum, medulla/pons), Clitoral gland, Epididymides, Kidneys, Liver, Mammary gland – Females, Mammary gland – Males, Ovaries with oviducts, Parathyroid glands, Pituitary gland, Penis, Prostate gland (dorsolateral and ventral), Sciatic nerve, Seminal vesicles with coagulating glands, Spleen, Stomach, Testes, Thymus, Thyroid, Uterus – cervix, Vagina, Gross abnormalities.
- Histopathological examination was restricted to all males and females in the control and high dose groups killed at term, all animals killed or dying during the treatment period and all abnormalities in all groups. The examination of the stomach was extended to parental male and female animals of the other dose groups, since treatment-related changes were observed in the high dose group.

Postmortem examinations (offspring):

SACRIFICE
- Pups killed for humane reasons or those that had completed the scheduled test period (Day 4 or Day 14 post partum) were euthanised by intraperitoneal injection of sodium thiopenthal.

GROSS NECROPSY
- All pups found dead in the cage or sacrificed for humane reasons 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 at Day 14 post partum were examined for external abnormalities and sex confirmation by gonads inspection. All pups with abnormalities were retained in 10% neutral buffered formalin.

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

Statistics:

Standard deviations were calculated as appropriate. For continuous variables, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopatholgical findings was carried out by means of the non-parametric Kolmogorov Smirnov test. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p < 0.05.

The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Reproductive indices:

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

Females:
Copulatory Index (%) = (no. of females with confirmed mating / no. of females cohabitated) × 100
Fertility Index (%) = (no. of pregnant females / no. of females cohabitated) × 100

Males and females:
Pre-Coital Interval = The number of nights paired prior to the detection of mating

Offspring viability indices:

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

Pre-natal loss was calculated as a percentage from the formula:
(no. of visible implantations - live litter size at birth) / no. of visible implantations × 100

Pups:
Pup loss at Day 0 post partum was calculated as a percentage from the formula:
((Total litter size - live litter size) / Total litter size) × 100

Post-natal loss at Day 4 post partum (before culling) was calculated as a percentage from the formula:
((Live litter size at birth - live litter size at Day 4 (before culling)) / Live litter size at birth) × 100

Post-natal loss at Day 13 post partum (after culling) was calculated as a percentage from the formula:
((Live litter size on Day 4 (after culling) - live litter size on Day 13) / Live litter size on Day 4 (after culling)) × 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was observed in animals of both sexes from all treated groups with a dose-related frequency and incidence (only occasionally in individual males and females dosed at 250 mg/kg bw/day) from the first few days of the pre-mating phase.

Unscheduled Deaths:
500 mg/kg bw/day: Female X1380041 showed hunched posture, dyspnoea, rales, piloerection, salivation and staining on the nose prior to humane sacrifice on Day 12 of the pre-mating phase. Female X1380045 did not show any clinical signs prior to death on Day 8 of the pre-mating phase.

750 mg/kg bw/day: Female X1380067 had dyspnoea and staining on the nose prior to humane sacrifice on Day 3 of the pre-mating phase. Female no. X1380071 exhibited salivation from Day 9 of the pre-mating phase up to death on Day 9 post partum.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

500 mg/kg bw/day: Two females (X1380041 and X1380045) were sacrificed for humane reasons or found dead on Days 12 and 8 of the pre-mating phase, respectively.

750 mg/kg bw/day: Two females (X1380067 and X1380071) were sacrificed for humane reasons or found dead on Days 3 of the pre-mating or Day 9 of post partum phase, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Very slight reductions in body weight (not greater than 10%), only occasionally significant at statistical analysis, were observed in the high dose males from Day 8 of the pre-mating phase. Reductions in body weight gain (up to -69%, significant at statistical analysis) were also observed in high dose males when compared to controls. This change was due to body weight losses occasionally observed in individual animals of this group. High dose males showed a statistically significant reduction in mean terminal body weight (-11.5%) when compared to the control group. These treatment-related reductions, due to their occasional nature and general low magnitude, were not considered to be adverse.

No significant changes in body weight and body weight gain were observed during the study in the treated females, when compared to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Slight reductions in food consumption (up to -18%) were observed in high dose males when compared to controls. Very slight, non dose-related reductions in food consumption were also observed in the males of the low and mid-dose groups (up to -10%). Slight, only occasionally statistically significant reductions in food consumption (up to -11%) were noted in high dose females during the study. This was also occasionally observed in the low and mid dose females.

The above reductions, more evident in the high dose animals, were not considered to be adverse, due to their low magnitude.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled Deaths:

500 mg/kg bw/day:
- Female X1380041 showed diffuse, moderate squamous hyperplasia associated with submucosal chronic inflammatory reaction in the non glandular region of the stomach, moderate perivascular and peribronchial oedema, moderate aggregation of alveolar macrophages and mild alveolar haemorrhage in the lungs. The pulmonary pathology was considered the factor contributing to the animals health status, caused by a possible misdosing.
- Female X1380045 did not show any relevant pathological changes. The cause of death could not be determined.

750 mg/kg bw/day:
- Female X1380067 had moderate mucosal ulceration in the non glandular region of the stomach and moderate haemorrhage in the muscle of the nasal cavity (muzzle/head). The cause of illness for this female could have been related to traumatic injury.
- Female X1380071 did not show any relevant pathological changes. The cause of death could not be determined.

Scheduled Sacrifice:
In the stomach, squamous hyperplasia associated, in most instance, with mucosal ulcer and submucosal inflammation (neutrophils and mononuclear inflammatory cell infiltrates) were noted in all high dose males and females, and in all males and 7/8 females dosed at 500 mg/kg bw/day (mid-dose). A few high dose males also showed dilatation of intestinal tract (duodenum, jejunum and/or ileum). Males and females receiving the test item at 250 mg/kg bw/day (low dose) did not show pathological changes in the stomach.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The copulatory indices were 100% for controls and all treated groups of both sexes. The number of copulation plugs was comparable between control and treated groups. All females mated, however, one female in the low and one in the mid-dose groups were found not to be pregnant.

The fertility indices in males and females were 90% for controls and animals dosed at 250 mg/kg bw/day, 87.5% for animals dosed at 500 mg/kg bw/day and 100% for those receiving 750 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at this dose level.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at this dose level.

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Apparent no food intake, cold to touch and small appearance were in general the clinical signs noted in control and treated pups prior to death, with higher incidence in a few litters from the mid and high dose level, which showed an increased litter size and a higher incidence of mortality from birth up to culling. Since no reduction in total litter size was observed after culling in these groups in comparison with the control, this change was not considered to be treatment related.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No differences of toxicological significance were recorded in relation to total litter size, live litter size and mean pup loss at Days 1, 4 and 13 post partum.

One female at 500 mg/kg bw/day (X1380047) showed total litter loss on Day 4 post partum.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No differences of toxicological significance were recorded in relation to mean pup weights at Days 1, 4 and 13 post partum. A slight not dose-related statistically significant decrease in mean pup weight and litter weight was observed in all treated groups in comparison with controls. This change was not considered to be treatment-related.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

AGD was found not to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No effect on retention of nipples was found.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Treatment did not affect organ weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No relevant findings were observed

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No effects were observed on thyroid weights or thyroid hormone levels.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

>= 750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at this dose level.

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Detailed data are provided in tabular form under 'Attached background material'.

Conclusions:

Some slight but treatment and dose-related effects (post-dose clinical signs in both sexes, slight reductions in body weight and food consumption) were observed in animals dosed at 500 and 750 mg/kg bw/day. These effects were not considered to be adverse, due to their low magnitude.

Treatment-related changes were also seen in the stomach of animals dosed at 500 and 750 mg/kg bw/day and/or intestines of some animals dosed at 750 mg/kg bw/day at macroscopic and microscopic examination. Changes in the stomach were ascribed to a local irritant effect and were not sufficiently severe to affect the sexual function and fertility, as well as the development of the offspring.

No significant changes were observed in animals dosed at 250 mg/kg bw/day.

No effects of toxicological relevance of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring were observed at any of the dose levels investigated. Therefore, the No Observed Adverse Effect Level (NOAEL) was considered to be 750 mg/kg bw/day for males, females and pups.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII, Section 8.7, of Regulation (EC) No. 1907/2006 (REACH).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Screening study

The toxic effects on Sprague Dawley rats after repeated dosing by oral route with fatty acids, C10-12, esters with polylactic acid, sodium salts (Dermosoft decalact, CAS No. 1312021-45-6, EC No. 700-937-1) as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring, were investigated in a study according to OECD guideline 421 under GLP conditions (Longobardi, 2021). All doses (250, 500 and 750 mg/kg bw/day) were administered orally, by gavage. The control group received corn oil. Males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 34 or 35 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post-partum, for a total of 42 to 55 days. The following investigations were performed on parental animals of all groups: mortality, clinical signs, body weight, body weight gain, food consumption, oestrous cycle evaluation (2 weeks before start of dosing, during pre-mating and mating phases, prior to necropsy), mating performance, thyroid hormones determination for parental males, litter data, post mortem macroscopic observations, organ weights and histopathological examination. Routine histopathological examination was performed only in control and high-dose groups and it included identification of the stages of the spermatogenic cycle in all male animals.

Two Group 3 (dosed at 500mg/kg bw/day) females were sacrificed for humane reasons or found dead on Days 12 and 8 of the pre-mating phase, respectively. The first death was caused by misdosing. The second female did not show any clinical signs prior to death. At post-mortem examination, thickened epithelium in the non-glandular region of the stomach was observed. Microscopically no relevant pathological changes that could be related to the cause of death were found. In addition, two Group 4 (dosed at 750mg/kg bw/day) females were sacrificed for humane reasons or found dead on Days 3 of the pre-mating or Day 9 of post-partum phases, respectively. The cause of the death of the first female could be related to traumatic injury, indicated by moderate haemorrhage in the muscle of the nasal cavity (muzzle/head). At post-mortem examination of the second female, red colour in brain, pituitary, thymus, lungs and jejunum mucosa and firm consistency in the heart were observed. Microscopically, this animal did not show any relevant pathological changes that could be related to the cause of death.

Salivation was observed in animals of both sexes from all treated groups with a dose-related frequency and incidence from the first few days of the pre-mating phase. Very slight but treatment-related reductions in body weight, only occasionally significant at statistical analysis, were observed in the males dosed at 750mg/kg bw/day. Due to their occasional nature and general low magnitude, these reductions were not considered adverse. No treatment-related changes in body weight and body weight gain were observed during the study in the treated females, when compared to controls. Slight but treatment-related reductions in food consumption were observed in the animals dosed at 750 mg/kg bw/day. Due to their low magnitude, these reductions were not considered to be adverse. No changes in relation to thyroid hormone levels in parental males were recorded. Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show any treatment-related intergroup differences. No treatment-related differences were observed for implantation sites, pre-implantation loss, pre-natal loss and gestation length of females between the treated groups and the controls. All pregnant dams gave birth between Days 22 and 23 post-coitum. High-dose males showed a statistically significant reduction in mean terminal body weight when compared to the control group.

No treatment-related changes were observed in absolute and relative organ weights of treated animals, when compared to controls. Treatment-related macroscopic findings were noted in the stomach of males and females receiving the test substance at and above 500 mg/kg bw/day. Thickened non-glandular region of the stomach was recorded in 10/10 males and 3/10 females dosed at 750 mg/kg bw/day and in 5/10 males and in 1/8 females dosed at 500 mg/kg/day. In addition, slight distention of intestinal tract (duodenum, jejunum, ileum, caecum and/or colon) was noted in a few high dose males. Treatment-related microscopic observations (squamous hyperplasia associated, in most instances, with mucosal ulcer and submucosal inflammation) were present in the stomach of both sexes of animals receiving the test substance at or above 500 mg/kg bw/day. In addition, a few high-dose males also showed dilatation of intestinal tract (duodenum, jejunum and/or ileum). Males and females receiving the test substance at 250 mg/kg bw/day did not show pathological changes in the stomach. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. There were no treatment-related observations in the testes of treated males when compared with control.

In conclusion, treatment-related changes were found in the stomach of animals dosed at 500 and 750 mg/kg bw/day and/or intestines of some animals dosed at 750 mg/kg bw/day at macroscopic and microscopic examination. Changes in the stomach were considered a local irritant effect and were not sufficiently severe to affect the sexual function and fertility as well as the development of the offspring. No significant changes were observed in animals dosed at 250 mg/kg bw/day. No effects of toxicological relevance of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring were observed at any of the dose levels investigated. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) for parental systemic toxicity and reproduction was considered to be 750 mg/kg bw/day, the highest dose tested.

Effects on developmental toxicity

Description of key information

Screening test, oral (OECD 421): NOAEL (systemic, rat, m/f) = 750 mg/kg bw/day; NOAEL (development) = 750 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII, Section 8.7, of Regulation (EC) No. 1907/2006 (REACH).

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

Screening study

The reproductive/developmental toxicity screening test summarised in detailed in section ‘Effects on fertility’ also investigated the development of pups (Longobardi, 2021). For details regarding study the design, please refer to the summary provided above. With respect to pup development, the following parameters were investigated: Clinical signs, anogenital distance, external and internal examination of pups at necropsy. In addition, thyroid hormone levels and thyroid weight were determined in 1 pup/sex and group randomly selected at Day 14 post-partum.

No differences of toxicological significance in total litter size, live litter size, mean pup loss, sex ratio and mean pup weights were observed among the treated dams and the controls at birth and on Days 1, 4 and 13 post-partum. There were no treatment-related clinical signs in pups at any dose level. No differences in the anogenital distance (normalised value), performed on Day 1 post-partum, were seen between control and treated groups both for male and female pups. No nipples were observed in male pups. Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post-partum did not reveal any treatment-related effect. No differences were observed in the weight of thyroid of treated pups, when compared to controls. No changes in relation to thyroid hormone levels in pups on Day 14 post-partum were recorded.

Based on the findings in pup development, the No-Observed-Adverse-Effect-Level (NOAEL) for developmental toxicity was considered to be 750 mg/kg bw/day, the highest dose tested.

Justification for classification or non-classification

The available reproduction/developmental toxicity screening test with fatty acids, C10-12, esters with polylactic acid, sodium salts (Dermosoft decalact, EC No. 700-937-1, CAS No. 1312021-45-6) does not warrant classification for reproductive or developmental toxicity according to Regulation (EC) No. 1272/2008 (CLP). However, since the study investigated reproduction/developmental toxicity only on screening level, ‘data lacking’ is selected in section 2.1 of the technical dossier.