Reaction mass of N-(2-hydroxypropyl)decan-1-amide and N-(2-hydroxypropyl)octanamide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

31 January 2013 - 17 June 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age/weight at study initiation: on the first day of treatment, the males were approximately 10 weeks old and had a mean body weight of 388 g (range: 335 g to 438 g) and the females were approximately 9 weeks old had a mean body weight of 236 g (range: 203 g to 270 g)
- Housing: the animals were individually housed, except during pairing and lactation, in polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 8 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 13 February 2013 to 09 April 2013.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The dose formulations were prepared according to the following process, as described in an homogeneity/stability study:
- a small amount of test item was melt using water bath at a temperature of +50°C,
- the vehicle was warmed at +50°C using water bath,
- the appropriate amount of melt test item was weighed in the preparation beaker and the corresponding amount of vehicle was added,
- the test item and the vehicle were mixed using magnetic stirrer in the water bath at +50°C during 10 minutes,
- the obtained suspension was stirred at ambient temperature at least 30 minutes.

No correction factor was applied.
The test item dose formulations were prepared on a weekly basis, stored at room temperature protected from light prior to use and delivered to the study room at room temperature and protected from light.

When not on the day of formulation preparation, test item formulations were warmed under magnetic stirring at +50°C using water bath during at least 30 minutes and then kept under magnetically stirring at ambient temperature for at least 30 minutes before daily delivery to the study room.

VEHICLE
- Choice of vehicle: good suspension in corn oil
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: GC-FID
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: assessed in homogeneity study (satisfactory results)
Stability: assessed in stability study (stable after 9 days at room temperature and protected from light)

Duration of treatment / exposure:

In the males:
− 2 weeks before mating,
− during the mating period,
− until sacrifice (at least 5 weeks in total),

In the females:
− 2 weeks before mating,
− during the mating period (1 week),
− during pregnancy,
− during lactation until day 5 post-partum inclusive,
− until sacrifice for females which had not delivered.

Frequency of treatment:

Daily

Dose / conc.:

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

Remarks:

Controls

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of a previous 2-week toxicity study. In this study, three groups of three male and three female Sprague-Dawley rats received the test item as a suspension in corn oil at 100, 300 or 1000 mg/kg/day bw for 2 weeks by gavage. There were no unscheduled deaths. Reduced mean body weight gain and mean food consumption were noted in males during the first week of treatment. Clinical signs were ptyalism in all test item-treated animals and hunched posture in two males and one female at the high-dose during the second week of treatment. There were no test item-related macroscopic findings.

- Animal assignment: computerized stratification procedure.

Positive control:

no (not required)

Observations and examinations performed and frequency:

The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating and mating periods (food consumption not during mating), and during gestation on days 0, 7, 14 and 20 p.c. and lactation on days 1 and 5 p.p.. The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until day 5 p.p.. At the end of the treatment period, Functional Observation Battery, motor activity and laboratory investigations (hematology and blood biochemistry) were carried out on five males and five females.

Sacrifice and pathology:

The males were sacrificed after at least 5 weeks of treatment and the females on day 6 p.p.. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs from five males and five females in the control- and high-dose groups, on liver, thymus, seminal vesicles and bone marrow from five males and/or five females in the low- and intermediate-dose groups and on all macroscopic lesions.

Statistics:

Body weight, food consumption and reproductive data :
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being
considered as homogenous) or by Fischer exact probability test (proportions).

Hematology and blood biochemistry:
A specific sequence was used for the statistical analyses of hematology and blood biochemistry data.

Organ weight:
PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01) according to a specific sequence

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ptyalism, considered of minor toxicological significance, was observed in all animals at 300 and 1000 mg/kg/day bw from the first or second week of treatment and in most of the animals at 100 mg/kg/day bw but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg/day bw but more at 1000 mg/kg/day bw were considered to be of limited toxicological significance. Incidental findings consisted of half-closed eyes, reflux at dosing, cutaneous lesion and abnormal growth of teeth.

Mortality:

mortality observed, treatment-related

Description (incidence):

Ptyalism, considered of minor toxicological significance, was observed in all animals at 300 and 1000 mg/kg/day bw from the first or second week of treatment and in most of the animals at 100 mg/kg/day bw but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg/day bw but more at 1000 mg/kg/day bw were considered to be of limited toxicological significance. Incidental findings consisted of half-closed eyes, reflux at dosing, cutaneous lesion and abnormal growth of teeth.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, mean food consumption at 1000 mg/kg/day bw was statistically significantly reduced in the first week of treatment when compared with controls (which correlated with a non-statistically significantly lower mean body weight gain at that time). It became similar to controls in the second week of dosing. This slight and transient effect was considered to be of limited toxicological significance. Mean food consumption at 100 and 300 mg/kg/day bw was not affected. Mean food consumption in females was considered to be unaffected by the test item treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

APPT values were not considered to be affected (one control data in males was higher than the others hence the shortened mean values in the test item-treated groups; there was no dose-relationship in females). For the slightly higher mean white blood cell counts when compared with controls, a relationship with the test item treatment was considered to be doubtful (no clear dose-relationship, individual values generally included in historical control data, and no statistical level).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mean cholesterol level was higher in test item-treated female groups in a dose-related manner, reaching statistical and toxicological significance at 300 and 1000 mg/kg/day bw. All individual values in both groups were included in historical control data and in absence of adverse correlates in the study, this was considered to be non-adverse. There was no dose-relationship in males, but an effect of the test item may be suspected in two males treated at 1000 mg/kg/day bw which had a high cholesterol level (2.3 and 2.4 mmol/L). Mean sodium concentration was also statistically significant higher in females treated at 1000 mg/kg/day bw when compared with controls. Males or other electrolytes in females were not affected and the variation was not severe. Therefore, this finding was not considered to be of toxicological significance. An effect of the test item treatment on mean albumin concentration at 300 mg/kg/day bw in both sexes was considered unlikely as there was no dose-relationship. The increase observed in triglyceride levels at 300 and 1000 mg/kg/day bw in both sexes was considered to be incidental as there was no statistical level reached for the group means and no dose-relationship seen in individual data. Moreover, the increase at 1000 mg/kg/day bw was due to isolated animals per sex only.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

An effect of the test item treatment in males and females at 300 and/or 1000 mg/kg/day bw on mean motor activity data was considered to be equivocal but of limited toxicological significance. The vast majority of the individual data were similar to what can usually be seen in this type of study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared with controls, the mean absolute and relative liver weights were higher in males and females given 1000 mg/kg/day bw and in males given 300 mg/kg/day bw, reaching statistically significant values in both sexes at 1000 mg/kg/day bw (p<0.01 except for the absolute weight in females where it was p<0.05). This correlated histologically with minimal hepatocellular hypertrophy and was considered to be test item-related. The mean absolute and relative thymus weights were lower in females given 1000 mg/kg/day bw, without reaching a statistically significant value. This correlated with minimal to slight atrophy in two females and was considered to be probably test item-related. The mean absolute heart weight was higher in males given 100 mg/kg/day bw. In the absence of a dose-related trend, any relationship with the test item was considered to be excluded. Other changes in the mean organ weights were considered to be part of the normal variation in rats and without relationship with the test item.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The thymus was reduced in one female given 1000 mg/kg/day bw correlating in this animal with a small weight (0.1200 g) and histologically with slight atrophy. A relationship with the test item was considered to be likely. Irregular color was seen in the lungs of 3/5 males given 1000 mg/kg/day bw. This correlated histologically with presence of mucus and inflammation (chronic) and/or alveolar macrophages. These changes were consistent with aspiration or regurgitation of the oily vehicle or test item during the technical gavage procedures.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were observed in the liver and the thymus.

Liver
Minimal hepatocellular hypertrophy was seen in 1/5 males given the test item at 300 mg/kg/day bw and 4/6 males and 2/5 females given
1000 mg/kg/day bw. This non-adverse change was considered to be test item-related and correlated with the higher weight noted at necropsy.
Other changes seen in the liver, including the minimal foci of subcapsular necrosis seen in 1/5 control females, 1/5 males at 300 mg/kg/day bw and
2/5 females at 1000 mg/kg/day bw were considered to be fortuitous and without any relationship with the test item.

Thymus
Minimal or slight lymphoid atrophy was seen in 2/5 females given 1000 mg/kg/day bw, correlating with the lower weight at necropsy. Any relationship with the test item was considered to be likely but non-adverse (low number of animals affected and low grades). No test item-related changes were seen at 100 or 300 mg/kg/day bw.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

See details below.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Parental toxicity (non adverse)

Key result

Critical effects observed:

not specified

Conclusions:

Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C18-unsatd. MIPA, according to OECD Guideline 422, in compliance with GLP. Groups of ten male and ten female Sprague-Dawley rats received the read across substance at dose-levels of 0, 100, 300 or 1000 mg/kg bw/day daily by oral (gavage) administration 2 weeks before mating, during mating, gestation and until up to Day 5 p.p. The concentration of the dose formulation was checked in study Weeks 1, 3 and 6. The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 p.c. and lactation on Days 1 and 5 p.p. The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 p.p. The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on Days 1 and 5 p.p. At the end of the treatment period, Functional Observation Battery, motor activity and laboratory investigations (hematology and blood biochemistry) were carried out on five males and five females. The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 p.p. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs from five males and five females in the control- and high-dose groups, on liver, thymus, seminal vesicles and bone marrow from five males and/or five females in the low- and intermediate-dose groups and on all macroscopic lesions. The pups were sacrificed on Day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs. The read across substance concentrations checked during the study were within an acceptable range of variations when compared to the nominal values (± 15%). There was no read across substance in control formulations. There were no read across substance-related deaths. Clinical signs consisted of ptyalism in all animals at 300 and 1000 mg/kg bw/day and in most of the animals at 100 mg/kg bw/day (minor toxicological significance), as well as hypoactivity, loud breathing, piloerection and/or round back observed in a few animals at 300 and 1000 mg/kg bw/day for a few days (limited toxicological significance). There were no relevant effects on mean body weight, mean Functional Observation Battery (FOB), as well as on mean hematology parameters in any group and sex. An effect of the read across substance treatment on mean motor activity data at 300 and/or 1000 mg/kg bw/day was considered to be equivocal but of limited toxicological significance. In males, mean food consumption at 1000 mg/kg bw/day was reduced in the first week of treatment only (23 g/male/day, vs. 27, p<0.001). This effect was considered to be of limited toxicological significance. Mean food consumption in males at 100 and 300 mg/kg bw/day and in females were not affected. In females, mean cholesterol level was higher than in controls at 300 and 1000 mg/kg bw/day (up to 1.9 mmol/L, vs.1.2, p<0.01) and considered to be non-adverse in absence of adverse correlates in the study. There were no relevant blood biochemistry findings in females at 100 mg/kg bw/day or in males. At histopathology at 1000 mg/kg bw/day, minimal hepatocellular hypertrophy correlating with higher mean liver weight was seen in the liver of both sexes (about +28% in males and +20% in females compared to controls, p<0.01 generally). In females, mild lymphoid atrophy was seen in the thymus of 2/5 females, correlating with lower mean weight at necropsy (about -22% from controls). At 300 mg/kg bw/day, only minimal hepatocellular hypertrophy was seen in the liver of a single male correlating with minor higher mean absolute weight in this group. All these microscopic findings were considered to be non-adverse (low number of animals affected and/or minimal to slight grades). There were no histopathological effects at 100 mg/kg bw/day. Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day (Bentz, 2013). 

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined repeated dose toxicity study with the reproduction / developmental toxicity screening

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

From February 04, 2021 to September 08, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted on 29 July 2016.

Deviations:

yes

Remarks:

Female animals were supplied in the weight range of 197-222 g and not 175-200 g, as indicated in the Study Protocol, while males were in the range of 211-246 g instead of 200-225 g. These deviations had no impact on the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data for this species and strain at ERBC.

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Animal supply and acclimatization:
A total of 102 Hsd: Sprague Dawley SD rats (45 males and 57 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy. After arrival, on 28 January 2021, the weight range for each sex was determined (211-246 g for males, 197-222 g for females) and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian. An acclimatisation period of approximately 4 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

- Animal husbandry:
The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C±2°C and 55%±15%, respectively; actual conditions were monitored, recorded and the records retained. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day. From arrival to mating, 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). 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). Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week. Drinking water was supplied ad libitum to each cage via water bottles, except in the case of urinalysis investigations. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at ERBC. Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

-Allocation to groups:
On the day of allocation all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. Furthermore, female animals that exhibited anomalies in the oestrous cycle were not allocated to the main groups. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. At allocation to the study, the body weight variation of animals did not exceed 20% of the mean weight of each sex. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed five per sex per cage. The cages were identified by a label and recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each group were distributed to minimise possible environmental effects and or contamination.

Route of administration:

oral: gavage

Details on route of administration:

The test item will be administered orally, by gavage. The oral route has been selected as it is a possible route of exposure of the test item in man.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC

Details on oral exposure:

The required amount of test substance was suspended in the vehicle. The formulations were prepared weekly or daily (concentrations of 10, 30 and 75 mg/mL), according to stability data from ERBC study No. A4126. Concentrations were calculated and expressed in terms of test substance as supplied. The test substance was administered orally by gavage at a dose volume of 10 mL/kg body weight. 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 in ERBC Study no. A4126 in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in ERBC validation protocol (r > 0.99; accuracy 80-120%; precision CV < 10%). In ERBC Study no. A4126, 28-hour stability at room temperature and 15-day stability at 2-8°C were verified in the range from 10 to 100 mg/mL. According to ERBC SOPs, suspensions are considered to be stable if concentration and homogeneity, after the defined period of storage, are still acceptable (80%-120% for concentration and CV < 10% for homogeneity). The proposed preparation procedure for the test substance was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) in ERBC Study no. A4126 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in ERBC SOPs for concentration (80-120%) and homogeneity (CV < 10%). Samples of the preparations prepared on Weeks 1 and 5 (last week with males and females) were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (80-120% for concentration and CV < 10% for homogeneity). Chemical analysis was carried out by the Analytical Chemistry Department. The software used for this activity was Empower® 2 Build No. 2154.

Duration of treatment / exposure:

- Males: Animals were dosed once a day, 7 days a week, for 14 days prior to pairing, through the pairing period and thereafter until the day before necropsy, for a total of 33/34 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

- Females: Animals were dosed once a day, 7 days a week, for 14 days 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 38 to 65 days). Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.

Frequency of treatment:

Once daily, 7 days/weeks

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 rats per sex per dose

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

- Mortality:
Throughout the study, all animals were checked early in the morning and in the afternoon each working day. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day. Severely debilitated animals were observed carefully.

- Clinical signs:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs was recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions. Observations were performed at the same time interval each day (approximately 5 - 10 minutes and 1.30 - 2 hours post-dose), the interval was selected taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals.

- Clinical Observations (Functional Observation Battery Tests):
Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination (ERBC SOP no. ANI/344). Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded. All observations were recorded for individual animals.

- Grip strength and sensory reactivity to stimuli:
Once during the study, towards the end of treatment (during Week 5 for males and Day 12 post partum for females with viable litters, where possible), 5 out of 10 males and 5 out of 10 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength (ERBC SOP
no. ANI/344). Measurements were performed using a computer-generated random order.

- Motor activity assessment (MA):
Once during the study, towards the end of treatment (during Week 4 for males and on Day 12 post partum for females with viable litters where possible), 5 males and 5 females were randomly selected from each group and the motor activity (MA) were measured (for approximately 5 minutes) by an automated activity recording device (ERBC SOP no. ANI/346). Measurements were performed using a computer-generated random order.

- Body weight - Parental animals:
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum.

- Food consumption:
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from Day 1 of dosing up to mating. Individual food consumption for mated females was measured on gestation Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.

- Blood collection for metabolomics: At the end of treatment period, 1 mL was taken from all surviving males and females, under conditions of food deprivation. Blood samples were collected (from the retro-orbital sinus in the males, from the abdominal vena cava in the females) and immediately transferred with a 1 mL pipette tip into an Eppendorf tube containing 10 µL of 10% Titriplex III solution. The tube was immediately closed and gently mixed 5-6 times by inverting it. Blood was kept cool in ice water (approximately 4°C) during the collection period of up to 20 minutes. To separate plasma, the blood was centrifuged at 4°C, 20000 g for 2 minutes. Blood cells and plasma were separated; 0.5 mL of the supernatant plasma layer were pipetted with a 1 mL pipette tip into the second labelled Eppendorf tube and covered with a N2 atmosphere. Tubes were closed with the lid which was wrapped with Parafilm-M, and frozen at -80°Cwithin at maximum 30 minutes after centrifugation, then stored at -80°C until despatch for additional investigations (metabolomics analysis) at an external laboratory. Results of the metabolomic analysis, the corresponding data and their interpretation are the responsibility of the Sponsor and are reported separately.

- Clinical pathology investigations
Blood collection was performed for hormone determination (0.8 mL) from all animals at termination under condition of food deprivation. Blood samples for haematology, clinical chemistry and coagulation were collected, at the end of treatment period, from the 5 males and 5 females (females with viable litters) of each group, under condition of food depriva- tion. No samples were taken for animals sacrificed for humane reasons.
Following haematology and coagulation paramaters were assessed: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count, platelets and prothrobin time. Clinical chemistry parameters assessed corresponds to : alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, triglycerides, bile acids, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G Ratio, sodium, potassium, calcium, chloride, inorganic phosphorus.

-Urinalysis (Only males randomly selected)
During the last week of treatment, individual overnight urine samples were also collected from the same animals selected for clinical pathology investigations (5 males/group, randomly selected). Before starting urine collection water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis. The measurements performed on urine samples are as follows: appearance, volume (manually recorded), specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood.
These parameters were analysed by Menarini Aution Max AX 4280/Aution Eleven AE 4020/Aution Sticks 10EA or ATAGO Refractometer (for Specific gravity), according to in- ternal procedures. The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for: epithelial cells, leucocytes, erythrocytes, crystals, spermatozoa and precursors, other abnormal components.

- Blood collection and thyroid hormone determination (T4 and TSH)
Blood collection for hormone determination was performed from all animals at termination.
Males
Blood samples (approximately 0.8 mL) for hormone determination were collected under isoflurane anaesthesia from the abdominal vena cava . The order of collection was equalised between groups.
Females
As a part of the necropsy procedure, blood samples (approximately 0.8 mL) for hormone determination was withdrawn from the abdominal vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.

- Immunoanalysis - Thyroid hormone determination (T4 and TSH)
Samples were assayed to determine the serum levels of Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA).

Sacrifice and pathology:

-Sacrifice/Euthanasia: Parental animals and those that had completed the scheduled test period were killed by exsanguinations under isofluorane anaesthesia. The animal sacrificed for humane reasons was killed with carbon dioxide. Parental males: The males were killed after the mating of all females (up to Day 35 of the study). Parental females: The females with live pups were killed on Day 14 post partum. One female with total litter loss was killed shortly after the occurrence of total litter loss. The females showing no evidence of copulation were killed 25 days after the last day of the mating session. The females which did not gave birth 25 days after positive identification of mating was killed shortly after

- Necropsy: The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.

- Organ weights: From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed: abnormalities, adrenal glands, bone marrow, aecum, clitoral gland, colon, duodenum, epididymides, eyes, femur, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes, mammary gland, nasal cavity, oessophagus, ovaries, parathyroid gland, pituitary gland, penis, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal column, spinal cord, skeletal muscle, splee, stomatch, testes, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina. The ratios of organ weight to body weight were calculated for each animal.
Tissues were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

- Histopathological examination: After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). A detailed qualitative examination of the testes was performed in all control and high dose group males killed at term. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted 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. Identification of the stages of the spermatogenic cycle was carried out.

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. The non-parametric Kruskal-Wallis analysis of variance 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 mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation was observed in 8 out of 10 males and 9 out of 10 females dosed at 300 and and in all males and females dosed at 750 mg/kg/day with a dose-related frequency (only occasionally in one individual females dosed at 100 mg/kg/day), from the first few days of the pre-mating phase up to termination.
Animals of the control group did not show any sign during the whole treatment period. Piloerection, pallor and swollen neck were also occasionally seen in 1 female dosed at 300 mg/kg/day (animal no. X1680059), while dyspnoea was observed in 1 male dosed at 750 mg/kg/day (animal no. X1680076). Due to the transient occurence and the low incidence, these signs were not considered treatment-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male from Group 3 (dosed at 300 mg/kg/day, no. X1680048) and one male from Group 4 (dosed at 750 mg/kg/day, no. X1680078) were found dead on Days 9 and 13 of the pre- mating phase, respectively. In addition, 1 female from Group 2 (dosed at 100 mg/kg/day, no. X1680033) was sacrificed for humane reasons on Day 22 of the gestation phase. Altogether, these mortalities were not considered treatment-related.
Animal no. X1680048 (300 mg/kg/day) showed salivation and dyspnoea in the days be- fore death. Macroscopically, dark red colour and incomplete collapse of lungs and clear fluid in the thoracic cavity were noted. At microscopic observation, diffuse oedema and haemorrhage of the lungs were observed. Death was considered to be related to misgavage.
Animal no. X1680078 (750 mg/kg/day) showed salivation in the days before death. Macro- scopically, single dark red area of the calvaria and dark red fluid in the cranial and thoracic cavity were observed. At microscopic observation, sub meningeal haemorrhage of the brain, alveolar and interstitial haemorrhage of the lungs, were noted. Death was considered to be related to a traumatic impact of the head following an accidental fall from the cage.
Animal no. X1680033 (100 mg/kg/day), was sacrificed for humane reasons on Day 22 of the gestation phase for difficulty in parturition. Piloerection and pallor were observed prior to sacrifice. No abnormalities were detected at macroscopic observation. At microscopic observation, unilateral cortical hypertrophy was noted. On the basis of the prolonged time without completing the parturition, impaired parturition was considered to be the reason for sacrifice. The gross and microscopic evaluation did not allow to establish the cause of impared parturition. In the absence of a dose-relation and other treatment-related effects, the difficulty in parturition observed in this animal is not considered to be treatment- related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight and body weight gain for male and female animals were generally comparable between the treated and control groups, before and during mating, during gestation and post partum periods.
Before pairing, on Day 15, females receiving 750 mg/kg/day showed a decrease in body weight gain (-91%) compared to the control group, while statistically significant body weight losses were seen in the males dosed at 300 mg/kg/day on Day 15 of mating phase. These isolated changes were followed by a regular growth of the animals. Due to their occasional occurrence and/or in the absence of a dose-relation, these changes were not considered treatment-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment in both gender during the study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were recorded.
The statistically significant differences recorded between control and treated animals (mean corpuscular haemoglobin concentration in males, reticulocytes and platelets in females) were not dose-related and/or of minimal severity, therefore they were considered to be incidental.
No changes were recorded for coagulation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were recorded.
A statistically significant decrease of total bilirubin (48%) was recorded in males dosed at 750 mg/kg/day. The decrease of bilirubin has no biological/pathological significance, especially in rats where normal values are low (mean historical control data is 0.04 mg/dL) and the low limit of historical control data is 0 mg/dL.

Endocrine findings:

no effects observed

Description (incidence and severity):

No treatment related changes were observed for thyroid hormone determination in parental males.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes were observed in parental males.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in the open arena (neurotoxicity assessment) did not reveal changes attributable to the test item, compared to controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in terminal body weight and organ weights when compared to the controls. The increase in relative mean liver weights (+10% for relative liver weight when compared to controls) and brain weight (+7% for relative brain weight when compared to controls) in high dose treated males were not correlated to any histopathological changes and, therefore, they were considered unrelated to treatment.
Any other organ weight variations were in the range of expected spontaneous changes in rats of the same age and considered unrelated to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic observations at the end of the treatment period. Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic observations at the end of the treatment period. Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment. There were no test item-related microscopic observations in the testis (stage aware evalu- ation on PAS-stained slides).

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in the open arena (neurotoxicity assessment) did not reveal changes attributable to the test item, compared to controls

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

neuropathology

organ weights and organ / body weight ratios

serum/plasma biochemistry

serum/plasma hormone analyses

urinalysis

Key result

Critical effects observed:

no

Please see tables in attachment section.

Conclusions:

Under the study conditions, the NOAEL for general toxicity was considered to be 750 mg/kg/day for males and females.

Executive summary:

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted in rat with the read across substance C8-18 and C18-unsatd. MIPA according to OECD Guideline 422, in compliance with GLP. Four groups of ten Sprague Dawley rats (males and females) were exposed by oral gavage to increasing concentrations of the test substance (0, 100, 300 and 750 mg/kg/day). All doses were administered by gavage at a constant volume of 10 mL/kg body weight. 0.5% aqueous solution of carboxymethylcellulose was used as a vehicle. According to the study design, males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 33/34 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 38 to 65 days. The following investigations were performed: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry in five randomly selected animals/sex/group), macroscopic observations, organ weights. Routine histopathological examination was performed in control and high dose groups (five randomly selected animals/sex/group). No mortality occurred throughout the study. Salivation was the only treatment-related clinical sign recorded in males and females at 300 and 750 mg/kg/day. Piloerection, pallor, swollen neck and dyspnoea were also noted in a single male and female animal at 300 and 750 mg/kg bw/day, respectively. These clinical signs were not considered to be treatment-related due to their transient occurrence and low incidence. Neurotoxicity assessment (removal from the home cage and observations in an open arena), motor activity, grip strength and sensory reactivity to stimuli did not reveal changes attributable to the test substance. Body weight and body weight gain of treated animals did not show differences throughout the study when compared to the control group. The food consumption was comparable in all groups. No adverse findings were recorded in clinical pathology investigations (haematology including coagulation parameters and clinical chemistry). Hormone analysis did not show any relation to treatment. Urinalysis in male animals did not reveal changes attributable to the test substance. Under the study conditions, the NOAEL for general toxicity was considered to be 750 mg/kg/day for males and females (Longobardi, 2022).

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From April 09, 1999 to May 28, 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

HanIbm:WIST (SPF)

Details on species / strain selection:

Recognized by the international guidelines as the recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: RCC Ltd Biotechnology & Animal Breeding Division CH-4414 Füllinsdorf / Switzerland
Acclimatisation: 7 days
Age when treated: 6 weeks
Body weight: males: 135-153 g (mean 144 g) and females: 115-133 g (mean 123 g)
Temperature: 21 +/- 3°C and relative humidity: 40-70%
Light period: 12 hour light/dark cycle
Diet: pelleted standard diet Provimi Kliba 3433 , ad libitum and water: community tap water, ad libitum

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

The test substance formulations were prepared weekly. The test substance was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C). Homogeneity of the test substance in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken during acclimatization. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics Division) according to a HPLC method supplied by the sponsor.

Duration of treatment / exposure:

28 days and the reversibility of treatment-related changes was assessed after a treatment-free 14 days recovery period.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

5 mL/kg bw
(group 1)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

5 mL/kg bw
(group 2)

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

5 mL/kg bw
(group 3)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

5 mL/kg bw
(group 4)

No. of animals per sex per dose:

Groups 1 and 4: 10 males; 10 females
Groups 2 and 3: 5 males; 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

In this subacute toxicity study, the test substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg bw/day for a period of 28 days. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg bw/day. These animals were treated for 28 days and then allowed a 14-days treatment-free recovery period after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. From the animals of the low and middle dose groups, livers were examined to establish a no-effect level.

Positive control:

-

Observations and examinations performed and frequency:

- Observations for mortality/viability were recorded twice daily. The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28; and once daily during days 29-42 (recovery). The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter. During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals. The test was performed approximately 1 hour after application. NB. The results of the Functional Observational Battery are presented under week 4.
- The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer. Body weights were recorded weekly during pretest, treatment and recovery and before each necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

- Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AGF 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded. Locomotor (decreased or increased) activity was measured quantitatively with Activity Monitor AM 1052 system (Benwick Electronic Equipment Design Manufacture, England). Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 15-minute intervals as well as the total activity of the measuring period.

- Blood samples for hematology (hematology, methemoglobin and coagulation) and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.

- The following organ weights were recorded on the scheduled dates of necropsy:Brain, Thymus, Spleen, Heart, Kidneys, Testes, Liver, Adrenals, Epididymides

The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

Sacrifice and pathology:

- after 4 weeks
- after 6 weeks (post recovery)
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist. All animals surviving to scheduled necropsy were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution:
- Adrenal glands
- Aorta
- Bone (sternum, femur including joint)
- Bone marrow (femur)
- Brain (telencephalon, cerebellum, pons)
- Cecum
- Colon
- Duodenum
- Epididymides (fixed in Bouin’s solution)
- Esophagus
- Eyes with optic nerve (fixed in Davidson’s solution)
- Harderian gland (fixed in Davidson’s solution)
- Heart
- Ileum, with Peyer’s patches
- Jejunum with Peyer’s patches
- Kidneys
- Larynx
- Lacrimal gland (exorbital)
- Liver
- Lungs (infused with formalin at necropsy)
- Lymph nodes (mesenteric, mandibular)
- Mammary gland area
- Nasal cavity
- Ovaries
- Pancreas
- Pituitary gland
- Prostate gland
- Rectum
- Salivary glands (mandibular, sublingual)
- Sciatic nerve
- Seminal vesicles
- Skeletal muscle
- Skin
- Spinal cord (cervical, midthoracic, lumbar)
- Spleen
- Stomach
- Testes (fixed in Bouin’s solution)
- Thymus
- Thyroid (incl. parathyroid gland)
- Tongue
- Trachea
- Urinary bladder (infused with formalin at necropsy)
- Uterus
- Vagina
- Gross lesions

Statistics:

- Group means were calculated according to the definition of any mean value using the individual values per animal and the number of animals.
- The following statistical methods were used to analyze grip strength, locomotor activity, body weights, organ weights and all ratios, as well as clinical laboratory data:
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher’s exact-test was applied to macroscopic findings.

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical signs were evident in any animal of any group during daily observation or during weekly observation (pretest and weeks 1-3). No test substance-related clinical signs were evident in any animal of any group during the Functional Observational Battery performed at week 4. Salivation, slight to moderate in degree, was observed in one male treated with 200 mg/kg bw/day (treatment days 18-23) and in up to three males treated with 1000 mg/kg bw/day (two males on treatment day 17, three males on treatment days 18-23). Slight salivation was noted in up to two females treated with 1000 mg/kg bw/day (two females on treatment day 17, one female on treatment days 18-19). In view of the infrequent occurrence of this finding, it was considered to be incidental. During pretest, slight hyperactivity was noted in one male foreseen for treatment with 200 mg/kg bw/day. Moderate hyperactivity was noted in one male and one female of the control group during week 3 of treatment. All other animals were without abnormalities.

Mortality:

no mortality observed

Description (incidence):

All animals survived the scheduled study periods.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance-related differences in body weight development were noted at any dose level.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A transient reduction of food consumption was noted at 200 mg/kg bw/day and 1000 mg/kg/day. When compared with the control and pretest values, test substance-related inappetence was noted in males and females treated with 200 mg/kg bw/day and 1000 mg/kg bw/day during treatment week 1. This finding was also noted in the latter group during treatment week 2. During subsequent weeks, food consumption compared favorably. No effects upon food consumption were noted in rats treated with 50 mg/kg bw/day. During the 14-day recovery period, the mean daily food consumption of males treated with 1000 mg/kg bw/day exceeded that of the controls. This was considered to be a compensatory reaction. The females treated with 1000 mg/kg bw/day consumed slightly less feed during the recovery period when compared with the control females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant differences of a few hematology parameters to control values were noted in males and females (higher leukocyte count in males treated with 1000 mg/kg bw/day, higher ratio of middle fluorescent reticulocytes in males treated with 200 mg/kg bw/day, higher absolute lymphocyte count in males treated with 1000 mg/kg bw/day, lower mean corpuscular hemoglobin and lower mean corpuscular hemoglobin concentration in females treated at 1000 mg/kg bw/day, shorter activated partial prothrombin times in females treated at 1000 mg/kg bw/day). None of the observed differences were supported by dose-response relationships, by similar findings in the opposite sex or by concomitant differences in related parameters, and therefore were considered to be incidental. All remaining differences to the control values at the end of the recovery period were also considered to be incidental.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A small number of test substance-related differences to the control values were noted. These differences were restricted to males and females treated with the test substance at 1000 mg/kg bw/day and included higher activities of alanine aminotransferase and alkaline phosphatase, higher phosphorus levels (1000 mg/kg bw/day), higher albumin levels higher albumin/globulin ratio. A number of statistically significant differences to the control values were noted. In the absence of dose-response relationships, similar findings in the other sex or concomitant changes in related parameters, the following differences were considered to be incidental:
Higher plasma glucose levels in males (1000 mg/kg bw/day), higher creatinine levels in males (1000 mg/kg bw/day); lower bilirubin levels in females (50 mg/kg bw/day and 1000 mg/kg bw/day), lower plasma cholesterol levels in males (1000 mg/kg bw/day), higher triglyceride levels in males (50 mg/kg bw/day) and females (200 mg/kg bw/day and 1000 mg/kg bw/day), higher phospholipid levels in males (50 mg/kg bw/day), lower activity of aspartate aminotransferase in females (200 mg/kg bw/day), lower lactate dehydrogenase in males (50 mg/kg bw/day) and higher lactate dehydrogenase levels in females (1000 mg/kg bw/day), higher calcium levels in males (1000 mg/kg bw/day), lower chloride levels in males (1000 mg/kg bw/day), increased total protein in males (50 mg/kg bw/day), higher globulin levels in males (50 mg/kg bw/day), lower albumin/globulin ratio in males (50 mg/kg bw/day). All remaining differences to the control values at the end of the recovery period were also considered to be incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 4 weeks: The absolute and relative liver weights of male and female rats treated with 1000 mg/kg bw/day were markedly higher than those of the controls. The differences were generally statistically significant and considered to be related to the treatment with the test substance. The slightly lower thymus weights in males treated with 200 mg/kg bw/day or 1000 mg/kg bw/day (thymus/body weight ratio) were not present in the females and these differences were considered to be incidental. All other organ weights were considered to be unaffected by treatment when compared with the test substance.

After 6 weeks: No differences of statistical significance were evident in the males or females when compared with the control animals. The higher liver weights noted in males and females treated previously with 1000 mg/kg bw/day were largely absent after recovery.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic findings attributed to treatment with the test substance were not evident during necropsy. All macroscopic findings recorded were unremarkable and within the range of spontaneous alterations which may be seen in rats of this age and strain. They consisted of renal pelvis dilation, reddish discoloration or discolored foci in several organs, incompletely collapsed lungs, thickened thyroid glands and dilated uterine horns filled with fluid.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Grip strength and locomotor activity: No test substance-related differences between groups were ascertained

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings which were test substance-related consisted of hepatocellular hypertrophy at minor degrees and hepatocellular cytoplasmic eosinophilia in both sexes treated with 1000 mg/kg bw/day. These findings were not accompanied by any inflammatory or degenerative lesion. There were no such findings evident in animals sacrificed after a 14-day recovery period.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

For detailed results tables and figures, kindly refer to the attached background material section of the IUCLID.

Conclusions:

Under the study conditions, the systemic NOAEL in rats was established at 200 mg/kg bw/day (equivalent to 188.2 mg a.i./kg bw/day).

Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, isoC18 MIPA (94.1% active), according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. The substance was administered daily by oral gavage to Wistar rats at dose levels of 0, 50, 200 and 1000 mg/kg bw/day for a period of 28 d. The groups comprised 5 animals per sex which were sacrificed after 28 d of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg bw/day. These animals were treated for 28 d and then allowed a 14 d treatment-free recovery period, after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during Week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post-mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. No test substance-related clinical signs were noted at any dose level. Body weights and food consumption were unaffected by treatment. Treatment-related findings were generally restricted to animals at 1000 mg/kg bw/day. These included higher activities of alanine aminotransferase and alkaline phosphatase, higher phosphorus levels, higher albumin levels and higher albumin/globulin ratio. Absolute and relative liver weights of animals at 1000 mg/kg bw/day showed test substance-related increases which were reversible after recovery. Liver changes consisting of hepatocellular hypertrophy at minor degrees of severity and eosinophilia of the hepatocellular cytoplasm were noted at 1000 mg/kg bw/day. These findings were considered to be caused by enzymatic changes and due to an adaptive metabolic response to a xenobiotic. The findings were resolved completely after the 14 d recovery period. Under the study conditions, the systemic NOAEL in rats was established at 200 mg/kg bw/day (Braun, 1999).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral (28 days)

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, isoC18 MIPA (94.1% active), according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. The substance was administered daily by oral gavage to Wistar rats at dose levels of 0, 50, 200 and 1000 mg/kg bw/day for a period of 28 d. The groups comprised 5 animals per sex which were sacrificed after 28 d of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg bw/day. These animals were treated for 28 d and then allowed a 14 d treatment-free recovery period, after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during Week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post-mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. No test substance-related clinical signs were noted at any dose level. Body weights and food consumption were unaffected by treatment. Treatment-related findings were generally restricted to animals at 1000 mg/kg bw/day. These included higher activities of alanine aminotransferase and alkaline phosphatase, higher phosphorus levels, higher albumin levels and higher albumin/globulin ratio. Absolute and relative liver weights of animals at 1000 mg/kg bw/day showed test substance-related increases which were reversible after recovery. Liver changes consisting of hepatocellular hypertrophy at minor degrees of severity and eosinophilia of the hepatocellular cytoplasm were noted at 1000 mg/kg bw/day. These findings were considered to be caused by enzymatic changes and due to an adaptive metabolic response to a xenobiotic. The findings were resolved completely after the 14 d recovery period. Under the study conditions, the systemic NOAEL in rats was established at 200 mg/kg bw/day (Braun, 1999).

Oral (combined repeated dose toxicity and reproductive developmental screening)

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted in rat with the read across substance C8-18 and C18-unsatd. MIPA according to OECD Guideline 422, in compliance with GLP. Four groups of ten Sprague Dawley rats (males and females) were exposed by oral gavage to increasing concentrations of the test substance (0, 100, 300 and 750 mg/kg/day). All doses were administered by gavage at a constant volume of 10 mL/kg body weight. 0.5% aqueous solution of carboxymethylcellulose was used as a vehicle. According to the study design, males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 33/34 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 38 to 65 days. The following investigations were performed: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry in five randomly selected animals/sex/group), macroscopic observations, organ weights. Routine histopathological examination was performed in control and high dose groups (five randomly selected animals/sex/group). No mortality occurred throughout the study. Salivation was the only treatment-related clinical sign recorded in males and females at 300 and 750 mg/kg/day. Piloerection, pallor, swollen neck and dyspnoea were also noted in a single male and female animal at 300 and 750 mg/kg bw/day, respectively. These clinical signs were not considered to be treatment-related due to their transient occurrence and low incidence. Neurotoxicity assessment (removal from the home cage and observations in an open arena), motor activity, grip strength and sensory reactivity to stimuli did not reveal changes attributable to the test substance. Body weight and body weight gain of treated animals did not show differences throughout the study when compared to the control group. The food consumption was comparable in all groups. No adverse findings were recorded in clinical pathology investigations (haematology including coagulation parameters and clinical chemistry). Hormone analysis did not show any relation to treatment. Urinalysis in male animals did not reveal changes attributable to the test substance. Under the study conditions, the NOAEL for general toxicity was considered to be 750 mg/kg/day for males and females (Longobardi, 2022).

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C18-unsatd. MIPA, according to OECD Guideline 422, in compliance with GLP. Groups of ten male and ten female Sprague-Dawley rats received the read across substance at dose-levels of 0, 100, 300 or 1000 mg/kg bw/day daily by oral (gavage) administration 2 weeks before mating, during mating, gestation and until up to Day 5 p.p. The concentration of the dose formulation was checked in study Weeks 1, 3 and 6. The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 p.c. and lactation on Days 1 and 5 p.p. The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 p.p. The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on Days 1 and 5 p.p. At the end of the treatment period, Functional Observation Battery, motor activity and laboratory investigations (hematology and blood biochemistry) were carried out on five males and five females. The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 p.p. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs from five males and five females in the control- and high-dose groups, on liver, thymus, seminal vesicles and bone marrow from five males and/or five females in the low- and intermediate-dose groups and on all macroscopic lesions. The pups were sacrificed on Day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs. The read across substance concentrations checked during the study were within an acceptable range of variations when compared to the nominal values (± 15%). There was no read across substance in control formulations. There were no read across substance-related deaths. Clinical signs consisted of ptyalism in all animals at 300 and 1000 mg/kg bw/day and in most of the animals at 100 mg/kg bw/day (minor toxicological significance), as well as hypoactivity, loud breathing, piloerection and/or round back observed in a few animals at 300 and 1000 mg/kg bw/day for a few days (limited toxicological significance). There were no relevant effects on mean body weight, mean Functional Observation Battery (FOB), as well as on mean hematology parameters in any group and sex. An effect of the read across substance treatment on mean motor activity data at 300 and/or 1000 mg/kg bw/day was considered to be equivocal but of limited toxicological significance. In males, mean food consumption at 1000 mg/kg bw/day was reduced in the first week of treatment only (23 g/male/day, vs. 27, p<0.001). This effect was considered to be of limited toxicological significance. Mean food consumption in males at 100 and 300 mg/kg bw/day and in females were not affected. In females, mean cholesterol level was higher than in controls at 300 and 1000 mg/kg bw/day (up to 1.9 mmol/L, vs.1.2, p<0.01) and considered to be non-adverse in absence of adverse correlates in the study. There were no relevant blood biochemistry findings in females at 100 mg/kg bw/day or in males. At histopathology at 1000 mg/kg bw/day, minimal hepatocellular hypertrophy correlating with higher mean liver weight was seen in the liver of both sexes (about +28% in males and +20% in females compared to controls, p<0.01 generally). In females, mild lymphoid atrophy was seen in the thymus of 2/5 females, correlating with lower mean weight at necropsy (about -22% from controls). At 300 mg/kg bw/day, only minimal hepatocellular hypertrophy was seen in the liver of a single male correlating with minor higher mean absolute weight in this group. All these microscopic findings were considered to be non-adverse (low number of animals affected and/or minimal to slight grades). There were no histopathological effects at 100 mg/kg bw/day. Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day (Bentz, 2013).

Additional considerations

Based on an in-depth analysis of the available information (see read-across justification in Section 13 of the IUCLID dataset), it is the hypothesis that a read-across within and across MEA, DEA and MIPA derived alkanolamides is scientifically plausible and justified. While there is at present no evidence for putting the read-across hypothesis in question, some limitations, predominantly related to the quality of individual endpoint studies (including quality of the test substance characterisations) and existing higher tier endpoint data gaps, are recognized. Accordingly, additional physico-chemical and toxicology data were generated in Tier 1 of a tiered testing programme to strengthen the toxicokinetic and toxicological link within and across the members of the DEA, MEA, and MIPA subcategories.  

 

In Tier 1, a series of bridging studies according to OECD TG 421 and 422 were conducted with a representative short- and a long-chain substance of each subcategory (i.e., DEA, MEA, and MIPA). Additionally, taking advantage of the bridging studies samples, metabolomics analyses were conducted to enhance the quality and quantity of data from a biological perspective.  

 

Overall, the results of the Tier 1 testing confirmed and supported the hypothesis of a similar toxicological profile within and across the different sub-categories. All investigated substances displayed in line with existing data a similar systemic toxicity profile with no observed repeated dose toxicity at the highest tested dose (i.e., NOAELs ≥ 700 mg/kg/day) and absence of reproductive or developmental toxicity. The absence of significant metabolome changes is in line with the Tier 1in vivofindings, and thereby further confirming the read-across hypothesis that there is no significant difference in terms of type and strength of effects within and across the FAA subcategories.  

 

In the present dossier update, proposed Tier 2 studies have been included with the aim to generate a complete set of higher toxicology data for a selected >1000 tpa substance (i.e., OECD TG 408/443/414 (rats)/414 (2nd species). The testing in Tier 2 will be conducted with C8-18 and C18-unsatd. DEA, the substance that is generally perceived to be the most critical from a toxicological point of view due to the proposed hazard classification of DEA. Additionally, a few minor non-significant metabolomic changes were noted for the investigated DEA-FAA substance (i.e., C16-18 and C18-unsatd. DEA), suggesting some type of biological activity, possibly explaining some findings in the 1000 mg/kg bw/day dose group in the dose-range finding study. These observations support the selection and recommendation to investigate a DEA-FAA substance as a ‘worst case’ for the FAA category in Tier 2.  

 

The strategy and status overview are detailed in the document entitled ‘ECHA-DIAP - FAA testing strategy summary – Nov 22’, attached in Section 13 of the IUCLID dataset.

Justification for classification or non-classification

Based on the results of a 28 d repeated dose oral toxicity study (OECD Guideline 407) with the read across substance, IsoC18 MIPA and C18-unsatd. DEA, and two combined repeated dose toxicity studies with C18-unsatd. MIPA and C8-18 and C18-unsatd. MIPA, the test substance does not require classification for this endpoint according to CLP (EC 1272/2008) criteria.