Fatty acids, C12-14, .alpha.-sulfo, disodium salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 421, GLP, oral administration via diet, rat: NOAEL for reproductive performance and fertility = 12,000 ppm (equals a mean substance intake for the entire exposure duration of 619 and 901 mg/kg bw for males and females, respectively)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 March - 31 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Rheinland-Pfalz, 21.03.2017

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 0018279326
- Content: 100.0 g/100 g - 0.7 g/100 g (water) = 99.3 g/100 g
- Expiry date: Dec 2019
- Purity test date: Dec 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Analytical verifications of the stability of the test substance in the diet for a period of 32 days at room temperature were verified before the study was initiated with a comparable batch
- The stability of these preparations was also demonstrated over a period of 35 days under ambient conditions.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The required quantity of test substance was weighed in a beaker depending on the dose group
and thoroughly mixed with a small amount of food. Then further amounts of food were added to this premix and thoroughly mixed for 1 minute. Afterwards, further amounts of food,
depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 10 minutes in a laboratory mixer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): with diet

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The test guideline requires the rat to be used as the animal species. This rat strain was selected
since extensive historical control data are available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Animals were free from any clinical signs of disease
- Age at study initiation: 11-12 weeks (males); 10 weeks (females)
- Housing: individually during study period (during overnight matings male and female mating partners were housed together; pregnant animals and their litters were housed together until PND 13); grouped (up to 5 animals per sex and cage) during pretreatment
- Diet: ad libitum (from the day of supply to the day before necropsy); ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: about 3 weeks

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical and for microbiological contaminants. The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required quantity of test substance was weighed in a beaker depending on the dose group and thoroughly mixed with a small amount of food. Then further amounts of food were added to this premix and thoroughly mixed for 1 minute. Afterwards, further amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 10 minutes in a laboratory mixer.

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight (about 16.00 h until 06.30 - 09.00 h of the following morning) for a maximum of 2 weeks
- A vaginal smear was prepared after each mating and examined for the presence of sperm.
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day (GD) 0
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) towards the end of gestation.

Analytical verification of doses or concentrations:

yes

Remarks:

LC/ESI-MS

Details on analytical verification of doses or concentrations:

At the beginning (during premating), once during gestation and once during lactation of the
study each 3 samples were taken from the lowest and highest concentration for potential
homogeneity analyses. These samples were used as a concentration control at the same time.
At the time points mentioned above additionally one sample from the mid concentration was
taken for concentration control analysis.

Averaged recovery rates ranged from 80 to 119 %.

Duration of treatment / exposure:

males: 35 days; females: 59 days
The duration of treatment covered a 5 weeks in-life period (males) including 4 days mating (mating pairs were from the same test group) as well as a 2-weeks in-life period (females), 4 days mating period, the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.

Frequency of treatment:

continuously (via diet)

Dose / conc.:

1 000 ppm (nominal)

Remarks:

for females during lactation: 500 ppm;
= mean intake of approx. 54 mg/kg bw/d (males), approx. 77 mg/kg bw/d (females)

Dose / conc.:

4 000 ppm (nominal)

Remarks:

for females during lactation: 2000 ppm;
= mean intake of approx. 208 mg/kg bw/d (males), approx. 292 mg/kg bw/d (females)

Dose / conc.:

12 000 ppm (nominal)

Remarks:

for females during lactation: 6000 ppm;
= mean intake of approx. 619 mg/kg bw/d (males), approx. 901 mg/kg bw/d (females)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: During the lactation period the test substance concentrations in the diet of the
F0 females were reduced to 50%. This dietary adjustment derived from historical body weight and food
consumption data maintained the dams at the desired target doses of the test item during this period of increased food intake.

For details regarding the dosing see table 2 in "Any other information on materials and methods"

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes (for mortality, any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice with the following exceptions for the females:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 10, 14 and 20.
• Females with litter were weighed on the day after parturition (PND 1), 4, 7, 10 and 13.
The body weight change of the animals was calculated from these results.
Body weight was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight/day: Yes
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-10, 10-14, and 14-20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION: No

OTHER:

Thyroid hormones (males only)
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving adult males at termination
- Parameters checked: Total thyroxine (T4), Thyroid stimulating hormone (TSH)

Oestrous cyclicity (parental animals):

For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Sperm parameters (parental animals):

Parameters examined in parental males:
testis weight, epididymides weight, prostate weight, weight of seminal vesicles with coagulating glands; histopathological examination of epididymides and testes

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes (Standardization of litters was not performed in litters with ≤ 8 pups)
- If yes, maximum of 8 pups/litter (4 pups/sex/litter as nearly as possible); excess pups were sacrificed under isoflurane anesthesia by decapitation, blood was sampled for determination of thyroid hormone concentrations and pups were examined externally, eviscerated and their organs were assessed macroscopically.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number (on PND 0) and sex of pups, stillbirths, live births, macroscopically evident changes, clinical symptoms (including gross-morphological findings; at least once daily), postnatal mortality (at least once daily), body weight (on PND 1 and PND 4 (before standardization), anogenital distance (AGD) (on PND 1), presence of nipples/areolae in male pups (on PND 13), blood thyroid hormone concentrations (on PND 4 (surplus pups after standardization) and PND 13 (one selected male and one female pup per litter), gross necropsy (at sacrifice (PND 4 (surplus pups after standardiation), PND 13))

GROSS EXAMINATION OF DEAD PUPS
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, on study day 35 after start of test substance administration
- Maternal animals: All surviving animals, on study day 59 after start of test substance administration

GROSS NECROPSY
- Special attention was given to the reproductive organs.

ORGAN WEIGHTS
- The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Epididymides
3. Ovaries
4. Prostate (ventral and dorsolateral part together, fixed)
5. Seminal vesicles with coagulating glands (fixed)
6. Testes
7. Thyroid glands (with parathyroid glands) (fixed)
8. Uterus with cervix

HISTOPATHOLOGY
- Fixation of the following organs was followed by histotechnical processing, examination by light microscopy:
1. Testis
2. Epidymides
3. Ovaries

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 (surplus pups after litter standardization) and 13 (remaining pups after litter standardization) days of age.
- These animals were subjected to postmortem examinations (macroscopic and microscopic (if required)) as follows: the pups were examined externally and eviscerated, and the organs were assessed macroscopically.
- All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
- Animals with notable findings or abnormalities were further evaluated on a case-by-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.

GROSS NECROPSY
- All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.

HISTOPATHOLOGY
- Thyroid glands/parathyroid glands of one male and one female pup per litter at 13 days of age were fixed in neutral buffered 4% formaldehyde solution for possible further processing.

Statistics:

see table 1 in "Any other information on materials and methods"

Reproductive indices:

Male reproduction data:
- The pairing partners, the number of mating days until vaginal sperm were detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
- mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods")

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
- mating index, fertility index (for formulas see "Any other information on materials and methods")

Offspring viability indices:

- viability index, survival index (for formulas see "Any other information on materials and methods")

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights/body weight gain of low- and mid-dose F0 parental males and females were comparable to the concurrent control values during the entire study period.
The mean body weights of the high-dose F0 parental males (12000 ppm) were below the concurrent control throughout the study although the difference did not gain statistical
significance. The mean body weights of the high-dose females (12000 ppm) were below the concurrent control throughout the study, the difference was statistically significant on GD 20 and PND 1 - 4 (about 6% and 10%, respectively).
The body weight change of the high-dose males was statistically significantly below the concurrent control values during in-life days 0 - 7 and 0 - 28 (about 98% and 45%, respectively).
The body weight change of the high-dose females was statistically significantly below the concurrent control values during GD 0 - 20 (about 18%).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the high-dose (12000 ppm) F0 males was consistently below the concurrent control, the difference was statistically significant during in-life days 0 - 7 (about
14%). Food consumption of the high-dose (12000 ppm) F0 females was consistently below the concurrent control, the difference was statistically significant during during GD 14 - 20 (about 9%).

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

In parental males (test groups 1, 2 and 3; 1000, 4000 and 12000 ppm) no treatment-related alterations of T4 and TSH levels were observed.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration was similar: 4.0 / 3.9 / 3.9 and 3.9 days in test groups 0 - 3, respectively.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The female/male mating index was 100% in all test groups
the female/male fertility index was 100% all test groups
The gestation index was 100% in all test groups

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

4 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOAEL

Remarks:

fertility and reproduction

Effect level:

12 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no reproductive toxicity observed up to and including the highest dose tested.

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean pup body weights of the high-dose pups were statistically significantly below the concurrent control values during PND 7 - 13 (12-13%). Accordingly, mean pup body weight
change of the high-dose male and female pups were statistically significantly below the concurrent control values during PND 7 – 13. Overall, the high-dose pups gained 14% less weight during lactation days 1 - 13.

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):

The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few pups showed spontaneous findings at gross necropsy, such as dilated ureter and dilated renal pelvis.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all
these findings were not considered to be associated to the test substance

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

In male and female pups at PND 13 no treatment-related alterations of T4 and TSH levels were observed.
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 100% / 99.2% / 93% and 97.1% in test groups 0 - 3.
The survival index indicating pup survival during further course of lactation (PND 4 - 13) was 100% in all test groups

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

4 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

no

Table 3: Mean body weights for male and female rats

Mean body weight [g]: Sex: Male- Phase: In-life

	0 ppm	1000 ppm	4000 ppm	12000 ppm
Day 0	371.1	370.2	368.7	368.5
Day 7	383.9	382.2	378.6	368.7
Day 13	388.8	387.8	380.7	375.4
Day 21	396.7	395.2	388.3	380.9
Day 28	404.3	402.7	395.5	386.9

 

Mean body weight [g]: Sex: Female- Phase: In-life

	0 ppm	1000 ppm	4000 ppm	12000 ppm
Day 0	224.6	222.8	221.8	223.4
Day 7	226.8	227.4	227.4	225.0
Day 13	234.1	234.1	228.8	228.0

 

Mean body weight [g]: Sex: Female- Phase: Gestation

	0 ppm	1000 ppm	4000 ppm	12000 ppm
Day 0	229.3	227.4	227.3	228.5
Day 7	256.4	253.8	254.7	253.0
Day 10	266.2	263.0	262.4	259.5
Day 14	284.0	279.0	278.2	273.8
Day 20	346.6	337.0	334.8	324.9 *

 

Mean body weight [g]: Sex: Female- Phase: Lactation

	0 ppm	1000 ppm / 500 ppm	4000 ppm / 2000 ppm	12000 ppm / 6000 ppm
Day 1	263.8	256.4	258.4	238.3 **
Day 4	276.3	272.6	272.6	259.9 *
Day 7	277.8	278.6	281.4	269.7
Day 10	296.5	293.8	293.5	282.5
Day 13	300.0	296.2	297.7	286.0

 

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01

Table 4: Summary Live pubs/litter

	0 ppm	1000 ppm / 500 ppm	4000 ppm / 2000 ppm	12000 ppm / 6000 ppm
Pups delivered	123	114	109	117
Found dead [pub]/Dead	0	0	0	2
Stillborn /Dead	1	4	1	1
cannibalized [pup] / Dead	0	1	6	0
Pubs surviving day 0 -4	122	109	102	114

From PND 5 - 13 no pub mortality was observed

Conclusions:

In this Reproduction/Developmental Toxicity Screening Test exposure to the test substance resulted in signs of systemic toxicity at a concentration of 12000 ppm, such as reduced food consumption and impaired body weight development. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 4000 ppm for male and female Wistar rats. The no observed adverse effect level (NOAEL) for fertility and reproductive performance was 12000 ppm, the highest tested dose. The no observed adverse effect level (NOAEL) for developmental toxicity was 4000 ppm, based on lower pup weights at 12000 ppm.

Executive summary:

In this Reproduction/Developmental Toxicity Screening Test, performed in accordance to OECD TG 421 and in compliance with GLP, the test substance was administered to groups of 10 male and 10 female healthy young Wistar rats (F0 animals) as a constant homogeneous addition to the food in different concentrations (0 ppm, 1000 ppm, 4000 ppm, and 12000 ppm).

Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was also demonstrated over a period of 35 days under ambient conditions. The overall mean dose of test substance throughout all study phases was approx. 54 mg/kg body weight/day (mg/kg bw/d) in the 1000 ppm group, approx. 208 mg/kg bw/d in the 4000 ppm group and approx. 619 mg/kg bw/d in the 12000 ppm group (males) and approx. 77 mg/kg bw/d in the 1000 ppm group, approx. 292 mg/kg bw/d in the 4000 ppm group and approx. 901 mg/kg bw/d in the 12000 ppm group (females).

The duration of treatment covered a 5 weeks in-life period (males) including 4 days mating (mating pairs were from the same test group) as well as a 2-weeks in-life period (females), 4 days mating period, the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.

Signs of general systemic toxicity were observed in parental animals exposed to 12000 ppm taking reduced food consumption and impaired body weight development into account. Reduction of food consumption and body weight gain was particularly distinct in parental females towards the end of gestation and during early lactation. Clinical pathology revealed no treatment-related adverse effects on thyroid hormones in any of the dose groups. Regarding pathology, all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility, reproductive performance and delivery were not impaired by test-substance up to a concentration of 12000 ppm, as was demonstrated by unchanged fertility, gestation and live birth indices of pups in all test groups. Viability and survival indices as indicators for pup mortality in lactation were also not significantly altered.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 4000 ppm for male and female Wistar rats. The no observed adverse effect level (NOAEL) for fertility and reproductive performance was 12000 ppm, the highest tested dose.

Pup weights (average 12-13% below control) and weight gain (average 14% below control) were significantly lower in in the high-dose group (12000 ppm). In addition, a higher number of runts was noted in this test group. Most part of this developmental delay can certainly be attributed to the distinct maternal toxicity which was evident by reduced food consumption/body weight gain of the dams, in particular towards the end of gestation and during lactation. However, independently of the primary or secondary nature of the reduced pup weights the low magnitude of the growth disturbance suggests that it is likely for the pups to make a recovery over time. Thus, this effect was considered to be treatment-related and adverse, but of low toxicological significance.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

619 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a reliable Reproduction/Developmental Toxicity Screening Test (reliability 1), performed in accordance to OECD TG 421 and in compliance with GLP, the test substance was administered to groups of 10 male and 10 female healthy young Wistar rats (F0 animals) as a constant homogeneous addition to the food in different concentrations (0 ppm, 1000 ppm, 4000 ppm, and 12000 ppm). Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was also demonstrated over a period of 35 days under ambient conditions. The overall mean dose of Fatty acids, C12-14, a-sulfo, disodium salts throughout all study phases was approx. 54 mg/kg body weight/day (mg/kg bw/d) in the 1000 ppm group, approx. 208 mg/kg bw/d in the 4000 ppm group and approx. 619 mg/kg bw/d in the 12000 ppm group (males) and approx. 77 mg/kg bw/d in the 1000 ppm group, approx. 292 mg/kg bw/d in the 4000 ppm group and approx. 901 mg/kg bw/d in the 12000 ppm group (females). The duration of treatment covered a 5 weeks in-life period (males) including 4 days mating (mating pairs were from the same test group) as well as a 2-weeks in-life period (females), 4 days mating period, the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals. Signs of general systemic toxicity were observed in parental animals exposed to 12000 ppm taking reduced food consumption and impaired body weight development into account. Reduction of food consumption and body weight gain was particularly distinct in parental females towards the end of gestation and during early lactation. Clinical pathology revealed no treatment-related adverse effects on thyroid hormones in any of the dose groups. Regarding pathology, all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility, reproductive performance and delivery were not impaired by test-substance up to a concentration of 12000 ppm, as was demonstrated by unchanged fertility, gestation and live birth indices of pups in all test groups. Viability and survival indices as indicators for pup mortality in lactation were also not significantly altered.

 

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 4000 ppm for male and female Wistar rats. The no observed adverse effect level (NOAEL) for fertility and reproductive performance was 12000 ppm, the highest tested dose.

Effects on developmental toxicity

Description of key information

OECD 414, GLP, oral administration via gavage, rat: NOAEL for maternal toxicity = 200 mg/kg bw/d; NOAEL for developmental toxicity = 800 mg/kg bw/d

 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 January 2021 to September 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Batch No. 0021238322/0021197214

Species:

rat

Strain:

Wistar

Remarks:

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

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: females: 9 weeks old, males 11 weeks
- Weight at study initiation: females 200-225 g, males at least 336.3 g
- Fasting period before study: No, exception: As a part of the sacrificial procedure, blood samples for clinical chemistry investigations were withdrawn from the abdominal vena cava of 14 females/group, under food deprivation.
- Housing: Before and after mating, the animals were housed no more than 5 of one sex to a cage in
clear polysulfone cages measuring 59.5×38×20cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese); nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week. During the mating period, the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages measuring 42.5×26.6×18.5cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese) with a stainless steel mesh lid and floor.
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei 4, 20019 Settimo Milanese (MI), Italy
- Water: ad libitum
- Acclimation period: appr. 2 weeks

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

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous solution of carboxymethylcellulose (CMC 0.5%)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was suspended in the vehicle. The formulation was prepared daily or up to 7 days (concentrations of 5, 20 and 80 mg/mL) according to stability data obtained from a
previous study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the formulations prepared during the study (the first and the last week of treatment) were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in ERBC SOP’s for suspension (85-115% for concentration and CV <10% for homogeneity).

Details on mating procedure:

- Impregnation procedure: cohoused
- Females were paired one to one in the home cage of the male and left overnight.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

All animals were dosed once a day from Day 6 through Day 19 post coitum.

Frequency of treatment:

daily

Duration of test:

All animals were killed on Day 20 post coitum.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 mated female rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Time of day for (rat) dam blood sampling: in the morning on day 20 post coitum

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

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

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting
from Day 0 post coitum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 p.c.
- Organs examined: ovaries and uteri, brain, adrenals, kidney, liver, thyroid (the thyroid was also examined microsopically)

THYROID HORMONE DETERMINATION:
please refer to 'Any other information on materials and methods incl. tables'.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

As a part of the sacrificial procedure, blood samples for clinical chemistry investigations were withdrawn from the abdominal vena cava of 14 females/group, under isofluorane anaesthesia and under condition of food deprivation. Additionally, thyroid hormone determination was performed.
- Volume collected: about 0.8 mL per animal

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Anogenital distance of all live rodent pups: yes

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by analysis of variance (one-way Anova). Inter-group differences were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data (verified by Bartlett’s test). Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5.

Indices:

Pre-implantation loss, Post-implantation loss, Total implantation loss

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Hairloss was occasionally recorded in all groups including controls and was considered incidental.
Salivation was occasionally observed during the last days of gestation in 5 out of 25 females of the high dose group and, althought the short appearance, this sign was considered treatmet related.
Other findings recorded in one mid-dose animal (cyphosis, emaciated and teeth missing) and one high dose animal (piloerection, rales, semiclosed eyes) were considered as incidental or related to in-life procedures and were, thus, deemed as minor clinical signs, not related to treatment with the test item.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A trend of decrease in body weight starting from Days 12 post coitum, and body weight gain starting from Days 9 post coitum was detected in the high dose group when compared to controls and was considered to be related to treatment.
No treatment-related differences in body weight or body weight gain were noted in the low and mid-dose treated groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Slight statistically significant reduction in food consumption was noted in the high-dose females from Day 9 to Day 20 post coitum. This reduction was considered related to the exposure of the test item, indicating a slight maternal toxicity.
No significant changes were noted in the low and mid-dose groups, when compared to controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

High dose animals showed an increase of alanine aminotransferase (38%) and a decrease of cholesterol (30%) and triglycerides (65%). The changes of alanine aminotransferase and cholesterol were within the range of expected biological variation and therefore considered to be incidental. The decrease of triglycerides was mostly due to the high triglycerides values recorded in control animals and in those receiving 50 (low dose) and 200 mg/kg/day (mid-dose). This is due to the gestational hypertriglyceridemia, therefore the findings observed were considered to be unrelated to treatment.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

No differences between control and treated animals were recorded. Triiodothyronine (T3) was statistically significantly lower than controls in high dose females (29%). Most of the individual values were within the range of the control group data, were judged to be due to individual changes or due to biological variation and no other related changes were recorded (thyroxine and/or thyroid stimulating hormone changes, histopathological findings), therefore the T3 decrease was considered to be incidental.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No differences were detected in the gravid uterus weight between control and treated groups.
A statistically significant decrease in terminal body weight, corrected maternal body weight (terminal body weight at necropsy minus gravid uterine weight) and corrected body weight gain (terminal body weight at necropsy minus gravid uterine weight, minus body weight at Day 6 post coitum) was noted in high females. These decreases were considered related to the reduced increment in body weight noted in this group during the whole treatment period, indicating a slight maternal toxicity.
No treatment-related differences were observed in the remaining groups compared to controls. There was no effect of on the absolute and relative (to brain) organ weights in treated females when compared to controls.
Any organ weight variations were within the range of expected variations in Wistar rats of the same age and considered incidental and unrelated to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At post mortem examination, no treatment-related macroscopic observations were noted in treated females when compared to controls.
Any macroscopic observations (alopecia) were within the range of expected spontaneous findings in Wistar rats of the same age considered incidental and unrelated to the test item.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Minimal follicular distension was found in the thyroid of one control animal. No treatment-related changes were noted in thyroid gland of all treated females when compared to controls. The microscopic observations corresponding to the macroscopic observations in control and treated females (alopecia) were consistent with those known to occur spontaneously in untreated Wistar rats of the same age and were considered incidental and unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The percentage of post-implantation losses was significantly increased in the mid dose group. Total implanation losses were significantly increased in all groups except for the control. These statistically significant variations in total implantation loss of treated groups were considered incidental since they were not dose related or depended on the pre-implantation loss that occur prior to treatment.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically increase in uterine death noted in the mid-dose group was considered not treatment related as mostly due to the loss of a single animal that showed signs of discomfort during the in vivo phase.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

A total of 7 females were found not pregnant at necropsy: 1 each in the control and low
dose groups, 2 in the mid-dose group and 3 in the high dose group. This finding can not be related to the treatment as dosing started on day 6 p.c..

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A total of 6 foetuses were small (bodyweight<2.7g): 2 in the control group, 1 in the mid-dose group and 3 in the high dose group. The mean foetal weights per dose group were not significantly different from the control.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related differences were noted in the mean values of the anogenital distance of foetuses of both sexes maternally exposed at all dose levels compared to the control group. The statistically significant increase (approximately 13%) noted in female foetuses of low dose group compared to the control was considered incidental since the difference was slight and without any dose-relationship.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Hindlimb with abnormal shape as bent were noted in one foetus of the control group and 2 foetuses of the same litter in the high dose group. These findings were not confirmed as skeletal alteration at the skeletal examination of foetuses.

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In this Developmental Toxicity Study exposure to the test substance resulted in signs of systemic toxicity at a concentration of 800 mg/kg bw/d in the pregnant rats, such as reduced food consumption and impaired body weight development. Thus, the no observed adverse effect level (NOAEL) for maternal toxicity was 200 mg/kg bw/d. The no observed adverse effect level (NOAEL) for developmental toxicity was 800 mg/kg bw/d.

Executive summary:

The effects of the test substance were investigated, after oral administration in female Wistar Hannover rats during pregnancy and embryo-foetal development, from gestation Day 6 through Day 19. Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each. The test item was applied at the following concentrations (CMC 0.5 % served as vehicle): test group 1 (control): 0 mg/kg bw/d, test group 2 (low dose): 50 mg/kg bw/d, test group 3 (mid dose): 200 mg/kg bw/d, test group 4 (high dose): 800 mg/kg bw/d.

Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All surviving females were caesarean-sectioned onDay 20 post coitum and subjected to post mortem examination. Blood collection for clinical chemistry and hormone determination was performed on Day 20 post coitum. Organ weigtht (brain, adrenals, kidney, liver and thyroid) was performed in all females. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, liver and uterusweights, foetalweight and sexwere recorded. The anogenital distance (AGD) in all live foetuses was recorded. All foetuses were examined for external abnormalities. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

No animal died during the study. Clinical signs (salivation) noted in few high dose females were considered to be treatment related. A trend of decrease in body weight and body weight gain was detected in the high dose females, together with a slight statistically significant reduction in food consumption when compared to controls, indicating a slight maternal toxicity. No treatment-related effects concerning clinical biochemistry investigations or thyroid hormone determination were noted. There was no effect of on the absolute and relative (to brain) organ weights in treated females when compared to controls.

No treatment related effects were seen in litter data and sex ratios. No treatment related differences were noted in the anogenital distance in both sexes. The incidences of foetuses or litters with external findings did not indicate test item-related effect. No skeletal malformations were noted in foetuses.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

800 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and OECD Guideline conform study

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

Key study

The effects of the test substance were investigated according to OECD Guideline 414 after oral administration in female Wistar Hannover rats during pregnancy and embryo-foetal development, from gestation Day 6 through Day 19. Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each. The test item was applied at the following concentrations (CMC 0.5 % served as vehicle): test group 1 (control): 0 mg/kg bw/d, test group 2 (low dose): 50 mg/kg bw/d, test group 3 (mid dose): 200 mg/kg bw/d, test group 4 (high dose): 800 mg/kg bw/d. No animal died during the study. Clinical signs (salivation) noted in few high dose females were considered to be treatment related. A trend of decrease in body weight and body weight gain was detected in the high dose females, together with a slight statistically significant reduction in food consumption when compared to controls, indicating a slight maternal toxicity. No treatment-related effects concerning clinical biochemistry investigations or thyroid hormone determination were noted. There was no effect of on the absolute and relative (to brain) organ weights in treated females when compared to controls. No treatment related effects were seen in litter data and sex ratios. No treatment related differences were noted in the anogenital distance in both sexes. The incidences of foetuses or litters with external findings did not indicate test item-related effect. No skeletal malformations were noted in foetuses. Based on these results, the NOAEL for maternal toxicity was determined to be 200 mg/kg bw/d and the NOAEL for developmental toxicity was determined to be 800 mg/kg bw/d.

Supporting study

In the Reproduction/Developmental Toxicity Screening Test, performed in accordance to OECD TG 421 and in compliance with GLP, the test substance was administered to groups of 10 male and 10 female healthy young Wistar rats (F0 animals) as a constant homogeneous addition to the food in different concentrations (0 ppm, 1000 ppm, 4000 ppm, and 12000 ppm). Signs of general systemic toxicity were observed in maternal animals exposed to 12000 ppm taking reduced food consumption and impaired body weight development into account. Reduction of food consumption and body weight gain was particularly distinct in parental females towards the end of gestation and during early lactation. Clinical pathology revealed no treatment-related adverse effects on thyroid hormones in any of the dose groups. Regarding pathology, all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. Pup weights (average 12-13% below control) and weight gain (average 14% below control) were significantly lower in in the high-dose group (12000 ppm). In addition, a higher number of runts was noted in this test group. Most part of this developmental delay can certainly be attributed to the distinct maternal toxicity which was evident by reduced food consumption/body weight gain of the dams, in particular towards the end of gestation and during lactation. However, independently of the primary or secondary nature of the reduced pup weights the low magnitude of the growth disturbance suggests that it is likely for the pups to make a recovery over time. Thus, this effect was considered to be treatment-related and adverse, but of low toxicological significance.

 

Justification for classification or non-classification

The available screening study is reliable and suitable for classification purposes under Regulation 1272/2008. No adverse effects on fertility were observed in a reliable screening study (OECD 421, GLP) up to and including the highest tested dose. In the same study, developmental toxicity in the F1 generation can certainly be attributed to the distinct maternal toxicity and it is likely for the pups to make a recovery over time. Thus, this effect was considered to be treatment-related and adverse, but of low toxicological significance. In the key study (OECD 414), no developmental toxicity of the test item after oral administration was revealed up to the highest dose tested (800 mg/kg bw/d). As a result, the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No 1272/2008, as amended for the seventeenth time in Regulation (EU) No 2021/849.